A 31-year-old man with left-sided testicular pain lasting a couple of months was referred to our urology department due to a suspected testicular tumor. Physical examination showed a hard, thickened, and small left testis on palpation with a diffuse, inhomogeneous ultrasonographic appearance. After a urologic examination, a left-sided inguinal orchiectomy was performed. The testis, epididymis, and spermatic cord were sent to pathology. Gross examination revealed a cystic cavity filled with brown fluid and the surrounding brownish parenchyma measuring up to 3.5 cm in diameter. Histologic examination showed a cystically dilated rete testis lined with cuboidal epithelium and a positive immunohistochemical reaction to cytokeratins. Microscopically, the cystic cavity was a pseudocyst filled with extravasated erythrocytes and abundant clusters of siderophages. The siderophages extended into the testicular parenchyma, surrounding the seminiferous tubules and spreading out around the ducts of the epididymis, which were also cystically dilated with siderophages inside their lumina. On the basis of clinical data, histological, and immunohistochemical analysis, the patient was diagnosed with cystic dysplasia of the rete testis. The literature shows an association between cystic dysplasia of the rete testis and ipsilateral genitourinary anomalies. Therefore, our patient underwent a multi-slice computed tomography scan, which revealed ipsilateral renal agenesis, a right seminal vesicle cyst reaching up to the iliac arteries, and a multicystic formation cranial to the prostate. Cystic dysplasia of the rete testis is a rare, benign cause of testicular mass, mostly observed in children. Around 60 cases have been recorded since the initial description in 1973 . Only twA 31-year-old male patient was referred to our Urology Department from an outside institution. He had been experiencing left-sided testicular pain for a few months and was suspected of having a testicular tumor. On palpation, the left testis was firm, thickened, and small. Ultrasonography showed a widespread, inhomogeneous appearance. A left-sided inguinal orchiectomy was performed. The spermatic cord, epididymis, and testis were sent to pathology. Gross inspection revealed a 3.5 cm-diameter cystic cavity with brown fluid content and the surrounding brownish parenchyma. Histologic examination showed a cystically dilated rete testis lined with cuboidal epithelium, positive for cytokeratins. Microscopically, the cystic cavity contained extravasated erythrocytes and large clusters of siderophages. The ducts of the epididymis were cystically dilated and had siderophages inside their lumina. Seminiferous tubules of the testicular parenchyma were also surrounded by siderophages . The diaThe precise etiology of cystic dysplasia of the rete testis is still unknown. One theory is that the mediastinum testis gradually degenerates into small cysts due to a lack of connection between the mesonephric duct and the germinal epithelium at the level of the rete testis . This thThe age at presentation of cystic dysplasia of the rete testis ranges from birth to 18 years, with a median of 5.2 years. Only two adult patients, aged 23 and 63 years, have been reported in the literature but were excluded from the review by Contini et al (


This study is to investigate the contrast sensitivity function (CSF) using the quick CSF (qCSF) test in Chinese adults with myopia.This case series study included 320 myopic eyes of 160 patients (mean age 27.75 ± 5.99 years) who underwent a qCSF test for acuity, area under log CSF (AULCSF), and mean contrast sensitivity (CS) at 1.0, 1.5, 3.0, 6.0, 12.0, and 18.0 cycle per degree (cpd). Spherical equivalent, corrected-distant visual acuity (CDVA), and pupil size were recorded.The spherical equivalent, CDVA (LogMAR), spherical refraction, cylindrical refraction, and the scotopic pupil size of the included eyes were − 6.30 ± 2.27 D (− 14.25 to − 0.88 D), 0 ± 0.02, − 5.74 ± 2.18 D, − 1.11 ± 0.86 D, and 6.77 ± 0.73 mm, respectively. The AULCSF and CSF acuity were 1.01 ± 0.21 and 18.45 ± 5.39 cpd, respectively. The mean CS (log units) at six different spatial frequencies were 1.25 ± 0.14, 1.29 ± 0.14, 1.25 ± 0.14, 0.98 ± 0.26, 0.45 ± 0.28, and 0.13 ± 0.17, respectively. A mixed effect model showed significant correlations between age and acuity, AULCSF, and CSF at 1.0, 12.0, and 18.0 cpd. Interocular CSF differences were correlated with the interocular difference of spherical equivalent, spherical refraction , and cylindrical refraction . The lower cylindrical refraction eye had higher CSF compared with the higher cylindrical refraction eye (0.48 ± 0.29 vs. 0.42 ± 0.27 at 12.0 cpd and 0.15 ± 0.19 vs. 0.12 ± 0.15 at 18.0 cpd).The age-related decrease in contrast sensitivity is at low and high spatial frequencies. Higher-degree myopia may show a decrease in CSF acuity. Low astigmatism was noted to affect the contrast sensitivity significantly. Contrast sensitivity function (CSF), evaluating the sensitivity thresholds at different spatial frequencies, is a more refined and sensitive parameter than visual acuity (VA) in evaluating visual-related quality of life and screening for diseases with a subjective visual impairment –4. The iMyopia is estimated to affect 50% of the world’s population by 2050 . Myopia The present study is a prospective cohort study, which included patients with myopia. The following were inclusion criteria: (i) patients aged ≥ 18 years; (ii) patients that had not used soft contact lenses for ≥ 2 weeks, or rigid gas permeable contact lenses for ≥ 4 weeks before the start of the study; (iii) patients with corrected-distant visual acuity  ranging from − 0.1 to 0.1. The exclusion criteria were patients with history of cataract, glaucoma, or other eye diseases and/or a history of systemic diseases.The study was approved by the ethics committee of Eye & ENT Hospital of Fudan University  and was conducted in accordance with the tenets of the Declaration of Helsinki. After obtaining written informed consent, patients were enrolled at the Eye & ENT Hospital of Fudan University, China, from November 2020 to July 2021.This study included 320 myopic eyes of 160 cases . Table Spherical refraction (nearsightedness or farsightedness), cylindrical refraction (astigmatism), and CDVA measurements were performed by optometrists using a phoropter , before and after the pupillary dilation. Pupil size  was measured using Nidek ARK-1(Nidek Technologies) under scotopic conditions. The qCSF test was conducted after the patients had corrected VA by wearing eyeglasses, according to the optometry results. Slit lamp examination and fundus examination were completed after pupillary dilation to exclude other eye diseases.2 and standard brightness 550 cd/m2, and contrast ratio of 4000:1. Sloan font was used for the numbers in the qCSF test  with a display area of 116.84 × 77.89 cm, resolution of 1920 × 1080, maximum brightness of 700 cd/mThe screen showed 25 stimuli and 3 numbers, each at different spatial frequencies. The stimuli were generated using a Bayesian adaptive procedure, and the digits were presented in fovea. A computer directly depicted the CSF curve with estimation and analysis of four parameters: peak CS (CSmax), peak spatial frequency (SFmax), bandwidth β , and low contrast intercept δ . The personalized stimuli were selected from a total of 2432 optotypes with 19 spatial frequencies and 128 contrast levels, depending on the participants’ response to the previous stimuli. At a sufficiently low contrast, the screen would appear uniform and unpatterned; at higher contrasts, the pattern would be visible , 16. Patp value of < 0.05 indicated statistical significance.Statistical Package for Social Sciences  was used for data analysis. Mean ± standard deviation were used for data expression; the Shapiro–Wilk test was used to evaluate data normality, and a mixed effect model was used for multi-level correlation analysis, in which the analyzed correlated parameters were independent of each other and the effect of inter-eye correlation was removed. Patients were grouped in the following two ways for the purpose of the study. First, the patients were categorized according to possible correlated factors: (i) age > or < 30 years ; (ii) cylindrical refraction > or <  − 2.0 DC; (iii) spherical refraction > or <  − 6.0 D; and spherical equivalent refraction  > or <  − 6.0 D. Second, eyes with higher values of spherical equivalent (closer to emmetropia), spherical refraction (lower-degree myopia), or cylindrical refraction (lower astigmatism) compared to the contralateral eye in each patient were placed in one group, and eyes with lower corresponding parameters were enrolled in another group. Age of 30 years was chosen as the demarcation point, because it serves to delineate young adults from middle-aged adults. Furthermore, spherical refraction was included to better describe the impact of the degree of myopia on qCSF readings, because spherical equivalent included both spherical refraction and cylindrical refraction. Spearman correlation analysis was performed to analyze correlation between differences in parameters of two eyes based on qCSF values. A generalized estimation equation test was performed to analyze between-group differences, interocular differences, and correlated two-eye data in normally distributed data, in which the analyzed correlated parameters were independent of each other and the effect of inter-eye correlation was removed. The correlation was analyzed between the differences of the correlated parameters and the interocular difference of CSF to elucidate the impact of these correlated parameters on qCSF readings. The Wilcoxon signed-rank test was used for non-normally distributed data. A Table The AULCSF, CSF acuity, and contrast sensitivity at high spatial frequencies of subjects < 30 years were significantly higher than those of patients aged > 30 years. However, no differences were found between patients grouped according to cylindrical refraction or SER Table .Table 3CTable In the present study, a qCSF test was performed on adults with myopia that had corrected VA. The distribution pattern of qCSF at different spatial frequencies and the possible influencing factors behind it were identified.2, while in patients with inherited retinal degeneration having decreased VA, it was more likely to be affected at high spatial frequencies. [A strong relationship between age and AULCSF has been previously reported.  The finduencies.  No retinThe difference in CSF between patients with macular degeneration and an age-controlled group at low luminance was less than that observed under standard luminance,  and the In contrast to previous reports that demonstrated no significant correlation between refractive error and CSF,  we foundThe interocular comparison suggested that eyes with lower cylindrical refraction tended to show higher CS at high spatial frequencies. This finding suggests the impact of interocular differences in cylindrical refraction on CS at high spatial frequencies. Therefore, a possible interference of image processing at different spatial frequencies cannot be ruled out; the image processing of interocular vision may be similar at low spatial frequencies but different at high spatial frequencies. The effect of cylindrical refraction on visual quality is certain.  InterestIn group comparisons Fig. , refractIn this study, considerable time had to be allotted for optometry and for prescription of eyeglasses. Therefore, additional studies are needed to compare the time taken to perform the tests for children and adults and to evaluate the characteristics of qCSF results without the influence of refractive correction. No feedback was provided (patients were not informed of whether their responses were correct or not) during the qCSF tests to reduce the time required for possible neurobiological learning, which may have interfered with our test results.The endpoint of manifest refraction is when adding + 0.25 DS to both eyes, it results in a slight blur of the patient’s best VA. An addition of − 0.25 DS will result in little effects, or it causes the letters to look darker and smaller. We chose patients with a LogMAR VA ranging from − 0.1 to 1.0 for this study. This range was set to minimize the effect of corrected VA on qCSF test results, because patients with a large difference of LogMAR VA may provide vastly different qCSF test results, under available VA correction. This specific inclusion criteria of the range of LogMAR VA could have helped in controlling the variables.This study has some limitations. First, peripheral CSF measurements were not included. A previous study found a similar CSF on the temporal and nasal sides,  and the In conclusion, the age-related decrease in CS was observed at low and high spatial frequencies. Higher-degree myopia may show a decrease in CSF acuity. Low astigmatism was noted to affect the CS significantly. Our findings have important implications in correction of optometric prescription under CSF guidance.


ATXN2, C9ORF72, FUS, SOD1, and TARDBP) and 13 controls were included in the study. Multipleanalytical approaches were employed, including metabolomic and metallomicsprofiling, as well as genetic screening, using CSF samples obtainedfrom the brain compartment. Data analysis involved the applicationof multivariate statistical methods. Advanced hyphenated seleniumand redox metal  speciation techniquesand nontargeted Fourier transform ion cyclotron resonance mass spectrometry-basedmetabolomics were used for data acquisition. Nontargeted metabolomicsshowed reduced steroids, including sex hormones; additionally, copperand manganese species were found to be the most relevant featuresfor ALS patients. This indicates a potential alteration of sex hormonepathways in the ALS-affected brain, as reflected in the CSF.Amyotrophic lateralsclerosis (ALS) is a lethal progressive neurodegenerativedisease, characterized by a loss of function of upper and lower motorneurons. This study aimed to explore probable pathological alterationsoccurring in individuals with ALS compared to neurologically healthycontrols through the analysis of cerebrospinal fluid (CSF), a medium,which directly interacts with brain parenchyma. A total of 7 ALS patientswith disease-associated mutations ( Dependingon studies’ accuracy, familial ALS covers ca. 5% of cases,with the majority of patients considered as sporadic because theyoccur randomly throughout the population.5 Several factors have been called into question in the genesis ofALS and the “multistep” pathogenetic model, which suggestsa view of the disease as a crossroad among genetic, neurometabolic,and environmental factors, represents a fascinating model of interaction,mostly accepted for many neurodegenerative diseases.7Amyotrophic lateralsclerosis (ALS) is a neurodegenerative diseaseof motor neurons; the loss of function of upper and lower motor neuronscauses progressive muscular atrophy and death usually within a fewyears after the diagnosis.9 a history of physicaltrauma/injury, electric shock, and previous exposure to organic solvents10 and pesticides12 were foundto be variably associated with the disease, supporting multifactorialand multipathway etiology of ALS.If genetic advances in the disease had revolutionized ALSunderstanding,environmental factors have been widely studied but frequently ledto conflicting results: which risk factors may contribute to diseaseonset and progression have not been identified so far. Among the moststudied environmental factors, exposure to heavy metals includinglead, mercury, and selenium,13 Up tonow, several such studies have been undertaken with respect to ALS.15 For instance, Kumar et al.16 used protonnuclear magnetic resonance (1H NMR) to examine 30 serumsamples from ALS patients, finding elevated quantities of glutamate,β-hydroxybutyrate, acetate, acetone, and formate and decreasedglutamine, histidine, and N-acetyl derivatives comparedto healthy controls (n = 25). Gray et al.13 used 1H NMR for profiling metabolitesin cerebrospinal fluid (CSF) samples of ALS patients (n = 41): glucose, lactate, citric acid, and ethanol were found asdiscriminating metabolites that are elevated in ALS compared to controls(n = 14). Wuolikainen et al.17 applied gas chromatography coupled to time-of-flight mass spectrometry(GC-TOF-MS) for the metabolite profiling of CSF; the authors reportedthat sporadic ALS patients had a heterogeneous metabolic signaturein the CSF, while familial ALS without superoxide dismutase-1 gene(SOD1) mutation was found to form a separate homogeneous group—glutamateand glutamine were reduced. Ultrahigh-performance liquid chromatography–MSwas used to describe CSF metabolites in ALS individuals by Blascoet al.18Metabolomics, that is, thestudy of the wide set of metabolites,in the biological media of ALS patients is a valuable tool for thestudy of disease pathology and a search for new potential biomarkersand therapeutic targets. The characterization of the metabolome allowsfor uncovering disease-specific signatures, which facilitates subgroupstratification and provides new insights into dysregulated biochemicalpathways in the affected tissue.20 steroid and steroid-like compounds,22 and so forth.Emerging research has highlighted the significant role that metalsand their disrupted homeostasis play in various neurodegenerativeconditions. For instance, aluminum or non-ceruloplasmin bound copper—theso-called free or exchangeable copper—was associated with thepathogenesis of Alzheimer’s disease or manganese with the developmentof Parkinson’s disease symptoms.24 Extensiveinvestigation on the potential association of metal exposure and metaldyshomeostasis with neurodegenerative disorders primarily centersaround evaluating metal levels in different biological samples . Nevertheless, the findings of thesestudies often yield conflicting results.25 It has been shown that the analysis of metals and their speciesin CSF provided a more stringent insight into the associations betweenmetal species changes and neurodegenerative conditions. Importantly,epidemiological studies mostly disregard the effect of metal species on the “classical”small organic molecule metabolites and vice versa. This is probablydue to the lack of instrumental capabilities to simultaneously conductmetabolomics and metallomics studies on the same set of samples. Individuallaboratories often specialize in either metabolomics or metallomics,leading to a potential limitation in comprehensive investigationsthat encompass both aspects. Metal species may affect different metabolicpathways involved in neurodegeneration, including, first, lipid peroxidationand inflammation, both influencing directly and through affectingprotein aggregation and enzymatic activity.29Different groups of metabolitesseem to be implemented in ALS pathology,including lipophilic compounds of different groups, e.g., membranelipids and metabolites of the arachidonic acid pathway,The current study aimedto investigate the small molecular weightmetabolites in the CSF of ALS individuals with disease-associatedgene mutations in comparison to matched controls and evaluate thepotential interplay between CSF metabolome and metallome for a betterunderstanding of the underlying pathological mechanisms. Particularly,selenium (Se) species appear to be either selenoproteins like glutathioneperoxidases (GPX), selenoprotein P (SELENOP), or thioredoxin reductases(TRXND), thus are direct actors in promoting neurodevelopment andprotection against oxidative stress, or they are Se-amino acids orinorganic Se species being shown to be detrimental in the frame ofALS. Metallic redox species also directly affect or even disrupt regularcell functions and can cause cellular dysfunction followed by lipidperoxidation, being in turn reflected in classical metabolomics studies.30 We hypothesized aninteraction between genetic and environmental/metabolic factors inthis instance, a topic so far not investigated in such patients butthat finds support from the current overall evidence about ALS etiology.31The case–control study was undertaken using the same setof CSF samples, which has been analyzed to examine the potential roleof Se and redox-active metals  in ALS.Supporting Information). All samples were collected solelyfor diagnostic purposes . To determinethe association of each —metabolite with the two samplegroups, orthogonal partial least square model discriminant analysis(OPLS-DA) was employed. Thepure concentration data of selenium, copper, iron, and manganesespecies in the CSF of ALS patients and controls have been reportedalready previously,x-axis)effectively distinguishes the samples into two distinct groups: controlsand ALS patients. The change in the identified metabolites for theALS individuals is shown in Supporting Information, Table S2).m/z) values assigned to hormones that exhibita stronger association with Fe and Mn and lipid oxidation product 10-nitrolinoleic acid (m/z 326.232), while they correlated with the concentrationof Se(IV) and relative (%) and absolute concentrations of Se(VI).For the ALS group, we observed a moderate correlation between sterollipid 3β-hydroxy-4β-methyl-5α-cholest-7-ene-4α-carbaldehyde(m/z 429.373) and relative contentsof SELENOP and GPX concentrations, respectively -basedchemical speciation approaches, particularly from seleno-metabolitedata since Se is a neuroprotective element of which, however, somecompounds were associated with ALS34 andthe data of redox-metal species, the latter associated with oxidativestress.35Theintegration of metallomics and metabolomics has been provento offer a profound understanding of the interrelationships betweenelements and metabolites at a molecular level. This approach has previouslybeen demonstrated in the analysis of CSF samples from patients withParkinson’s disease.18 This complicates the comparison of differentstudies and the translation of the metabolomics approach to clinicalapplications, making the use of an adequate statistical evaluationindispensable.Importantly, the wholeset of metabolites in the CSF is way toodiverse to be fully detected using a single technique, thus, comprehensivemetabolomics studies are based on the combination of several techniquessince some groups of metabolites are more suited for some techniques;the use of all available techniques in the same study is not possibledue to financial, technical, and analytical constraints.SOD1, FUS, and TARDBP genemutations and C9ORF72 and ATXN2 expansion,where we already found an increased SeMet content in the CSF30 as well as the potential role of copper.4 Altogether, these findings showed a biologicalagreement on a possible mechanism of toxicity related to ALS, namely,an impairment of protective mechanisms against oxidative stress, prooxidant,mitochondrial damage, and lipid peroxidation.In this study, we examined CSF samples from patientscarrying 36 It is also likely that the metabolic changes,induced by toxin exposure within the brain, are reflected in the CSF.38 With this further study, we aimed at evaluating the interactionsbetween metals and metabolites in ALS patients and controls in orderto identify new pathogenetic elements and possible therapeutic targets.To this last point,the use of a rarely available matrix, suchas CSF, in this study represents a strong suit. Metals and metabolitescan be found in altered concentrations in various human biofluids, and among these, CSF, due to its proximityto the central nervous system and relative poverty of other elements,is particularly suitable for studying neurodegenerative diseases.39 indicatingpotentially reduced brain metabolism in ALS reflected in CSF. Theassigned metabolites showed certain alterations in the CSF of ALSpatients, especially, steroids  as well as selenium and redox-activeselenoproteins  maybe involved. Cu, being the key element in activated ceruloplasmin,is known to be essential for oxidizing the pro-oxidative Fe2+ to redox-inactive Fe3+, thus reducing potential ROS generation.Manganese found to be associated with ALS samples  through oxidative-stress-mediatedmechanisms, samples 3, in tursteroids 2A as welsteroids 2C. Never48 A study of sex hormones in males and females may be an intriguingperspective of further insight into the role of steroids in ALS pathology.In this study, the number of specimens was too small to test for anysex differences.As far as steroids are concerned, they areknown to be implicatedin ALS as neuroprotective and neurotrophic agents acting through theirnuclear receptors or independently from them. Steroids include a widevariety of compounds including sex hormones that have been claimedas responsible for sex differences in ALS.50 the compounds that were found to be affected in the CSF of ALS individualsin the current study. Estrogens can exert neuroprotective activitybased on antioxidant activity, interaction with mitogen-activatedprotein kinase, cyclic AMP pathways, and with the activity of NF-κB,and mediate cytoskeleton dynamics through interacting with Rho-GTPasesand NADPH oxidase complex.51 Glucocorticoidcompounds, like 21-aminosteroids, have high antioxidant power andprevent lipid peroxidation. They increase GFAP expression, suppressnitric oxide by decreasing NADPH-diaphorase, and attenuate the aberrantexpression of both GAP-43 protein and mRNA in Wobbler motoneuronsthat are related to denervation and muscle atrophy.52Among steroids, a neuroprotective role hasbeen attributed to estrogens,aminosteroids, testosterone, and progesterone,Drosophila melanogaster.53Although there are a few human studies on ALS andsteroids, severalshreds of evidence come from different animal models of the disease.In Drosophila models, decreased expression of TDP-43 determined thecytoplasmic accumulations of the ecdysteroid receptor (EcR) and afailure to switch EcR-dependent gene programs from a pupal to adultpattern sustaining the hypothesis that TDP-43 loss of function causesneuronal loss due to defective steroid receptor-mediated gene programswitching in 54 Spinal motor neurons displayed a high densityof nuclear androgen receptor (AR), estrogen receptor (ER)α,ERβ, and progesterone receptor that was differentially expressedby sex,55 and nandrolone treatment markedlyenhanced motoneuron loss and astrocytic activation.56 Also, after orchiectomy,57 whereasprogesterone treatment delayed the disease progression, reduced mutantSOD1 protein levels and motoneuronal death through activation of autophagywere observed.49Functional studies in the SOD1G93Atransgenic mice showed thatestradiol delays disease onset and progression and increases survival.50 Also, treatment with allopregnanolone alleviated the alterationof several markers of neurodegeneration  and improved muscle performance.58 On the contrary, the same administration of nontargetedglucocorticoids in mice did not alter disease progression, maybe dueto poor CNS delivery. In fact, CNS-targeted, liposomal-encapsulatedglucocorticoid inhibited CNS inflammatory response, reduced motorneuron loss, and T2 hyperintensity on MRI of SOD1G93A mice.59 This further highlights the importance of studyingfluids in proximity to the CNS, given the high impermeability of theblood–brain barrier that makes serum and plasma determinationnot always representative of CNS pathomechanisms.Another animal model(Wobbler mice) showed a hypothalamic–pituitary–gonadalhypoactivity, and testosterone treatment induced a reduction of AR,ERα, and aromatase and an increase of Sigma-1 receptor mRNAs,with a reduction in neuroinflammation and oxidative/nitrosative stresstogether with a slowing down the disease progression.22 Other studiesshowed increased levels of cortisol60 andprogesterone61 in ALS patients with respectto controls, but this difference appeared to be too small to be usedas biomarkers. The studies, addressing CSF samples and patients withgene mutations associated with ALS, are lacking. Importantly, casesand controls at the time of the lumbar puncture (LP) did not takesteroidal treatment in our study. Unfortunately, the size of the ALSgroup, in the current study, does not allow for properly investigatingsexual dimorphism.The resultson sex hormones and other steroids (a putative depressionof their metabolism in ALS brain) raise the intriguing possibilityof a common pathway across different genetic mutations characterizedby steroids impairment, in which receptor up-regulation may correspondto an endogenous protective mechanism, and that could be targetedby available treatments like steroid hormones. In humans, mainly serumsteroids have been studied in ALS patients, showing increased levelsof testosterone in comparison with healthy controls, which did notdecline with aging differently from controls, and that was correlatedto disease progression.28 In general, steroids maysignificantly modulate redox balance in the body,29 which may involve redox-active metals and selenoenzymeslike GPX and TRXNRD. OPLA-DA study of the potential association ofmetal species and steroids showed that N--ethanolamide(m/z 350.305) was associated withtotal Mn (m/z 293.247) associated with unidentifiedMn species (Mn-unknown) and relative content of Fe(III) for the controls.Additionally, for the controls, some association of these Mn and Fespecies was observed for female sex hormone 2-methoxy-17β-estradiol(m/z 303.195), male steroid hormoneandrost-4-ene-3,17-dione (m/z 287.201),as well as other steroid species 3β,17β-dihydroxyetiocholane(m/z 293.247). Unfortunately, theliterature contains only scarce results on the interplay of steroidsand metal species.Interestingly, steroid hormones  were shown to affect brain metal levels(copper and zinc) and metal-binding metallothionein expression inmouse brains.total Mn 2for the21 so the role of the mentionedgroups of lipophilic compounds should warrant further lipidome-targetedstudies. The involvement of lipidome in ALS pathology in the currentstudy is partially supported by the findings related to selenoenzymesGPX and TRXND, which are involved in antioxidant protection, includingcounteracting lipid peroxidation and ferroptosis.64 Another supportive argument is the potential implicationof such redox-active metals as manganese and copper in ALS pathology(Z)), m/z 703.575) and fatty acid acyl glycoside corchoionosideC (m/z 387.201) in the ALS cases.Interestingly, sphingomyelins, including SM(d18:0/16:1(9Z), were shown to be discriminatory metabolites in the Cu-chelatorcuprizone-induced murine model of multiple sclerosis.65 Cell culture research also indicated the effect of Cu onsphingomyelins.66 ALS patients showed apositive correlation between copper status (blood plasma copper) andHDL–cholesterol, whereas in matched control, the associationwas found for both HDL- and LDL–cholesterol.67 Moreover, copper was found to be associated with oxidativestress of lipidic components in the rat astrocyte model of ALS.68 There is some evidence on the implication ofiron in membrane lipid metabolism too,69 through lipid peroxidation and ferroptosis.45 Our data . Then,we could not evaluate the influence of individual factors like sexor age on the observed results. In addition, the body weight indexwas not available for analysis. Further, only very small sample volumesper sample were available. Therefore, after the metallomics and nontargetedmetabolomics analysis of the CSF samples, the quantities of the remainingsamples (both ALS and control groups) were too low and running shortto confirm the identity of the compounds using targeted MS/MS analysis,which is a principal limitation of the current study.Ourstudy is subject to several limitations:one significant limitation is the small sample size of the geneticALS cohort, which is due to the rarity of ALS cases with confirmeddisease-related mutations. This constraint hampers the ability toconduct subgroup analyses, such as examining the impact of specificmutations since differences in the metallome/metabolome of each mutationwould be possible  as well as distinguish between disease-causing and disease-modifyingmutations , confirmed by genetic screening, and from matchedcontrol individuals. Finally, the entire data were evaluated usingadvanced statistical methods.Inthis study, we successfully showcased the effectiveness of integratingmetabolomics and metallomics in investigating potential pathologicalalterations within the CSF of individuals with ALS compared to neurologicallyhealthy controls. Through multivariate analysis, we were able to setthe strongest relations between the two different sets of data experiments,confirming already some knowledge discussed previously in the literatureand opening new scenarios for possible new hypotheses.Reducedsteroids, including sex hormones, as well as copper andmanganese species were found to be the most prominent features ofALS CSF samples. This indicates a potential impairment of sex hormonepathways in the ALS-affected brain, as reflected in the CSF. The findingsof the current study need to be further elaborated in larger cohortswith targeted studies, this corresponds, first of all, to the involvementof CSF lipidome in the ALS pathology.30 In brief, the CSF specimenswere provided by three major Italian ALS Referral Centers  from Italian patients. The patients were diagnosedwith definite or probable ALS since 2002 and were identified to carryan ALS-related gene mutation. All patients underwent LP at diagnosis.70 After performing all necessary clinical chemistry,trace element analysis, and chemical speciation, only the samples,which are not contaminated with blood and still have at least 0.5mL of CSF available, were enrolled for this study. The demographiccharacteristics of ALS patients are summarized in Supporting Information). The study was approved by the AziendaOspedaliero Universitaria of Modena Ethics Committee .We analyzed metabolites in CSF samplesof 7 ALS cases with disease-related mutations, assessed through genescreening 1,and 13C9ORF72 gene expansion was performed by repeat primed polymerasechain reaction(PCR) Amplidex PCR/CE C9ORF72 Kit .PCR and genetic sequencing were used to detect SOD1, VCP, TARDBP, and FUS gene mutations .72TUBA4A and ATXN2 gene analyseswere performed as previously reported.74 Control samplescame from patients who underwent LP due to a suspected neurologicalcondition that was later unconfirmed after complete clinical and instrumentalinvestigations based on the criteria proposed by Michalke et al.38Search for 4 In brief, after localizing L3–L4 interspace, skin swabs,an antiseptic solution, and an adhesive sterile drape were used tocreate a sterile field on the patient. CSF was withdrawn through a20 gauge needle after a local anesthetic, and 6–8 mL of CSFwere initially collected in sterile polypropylene tubes. The sampleswere immediately frozen and stored at −80 °C. To maintainblindness, all collected specimens were subjected to a rigorous labelingsystem. Any clinical or demographical data were removed. Anonymizedsamples were transported by air courier on dry ice to the HelmholtzZentrum München and kept continuously frozen at −80°C until use.CSFsamples were obtainedthrough LP, according to an established procedure at the three aforementionedNeurology Centers, as previously reported.l-argininefrom Sigma-Aldrich , and formicacid. Chemicals used for metallomics study werealready described in previous reports.75The chemicals used inthis study were as follows: methanol , 76 In brief, frozen CSFsamples were thawed on ice and vortex-mixed before treatment. C4 ZipTipSPME cartridges from Agilent Technologies  wereused for matrix separation and desalination of the CSF samples. 50μL of CSF was taken and diluted with 50 μL of purifiedwater (MilliQ). The cartridges were preliminarily primed with methanoland 2% formic acid in water. The diluted sample (100 μL) wasloaded onto the cartridges (on ice), rinsed with 2% formic acid solution,and eluted with 100 μL of methanol (on dry ice). All extractswere diluted in methanol 1:5 (v/v %) in methanol before analysis .Prior to direct input (DI) FT-ICR-MSanalyses, a solid-phase microextraction (SPME) was used based on aprotocol previously reported.33 An l-argininesolution (3 mg/L in methanol) was used for the external calibrationof the mass spectra with errors below 0.1 ppm. All measurements wereundertaken in positive ionization mode and ion accumulation time of300 ms was applied in the argon-filled collision cell for enhancedsensitivity. The resolution was on average R = 400,000at m/z 400 with a recorded timedomain transient size of 4 MW and length >1.6 s for each FT-ICR-MSacquisition, enabling an excellent signal differentiation on a molecularlevel. ESI source parameters were set as follows: an injection flowrate of 2 μL/min, a source operating temperature of 180 °C,a nebulizer gas flow rate of 2 L/min, and a dry gas flow rate of 4L/min. The spectra were recorded in an m/z range of 147–1400 Da. For the generation of eachmass spectrum, 300 scans were acquired.The measurementof metabolicprofiles was performed using an ultrahigh-resolution direct infusionFT-ICR-MS spectrometer SolariX ,equipped with a 12-T superconducting magnet  and an electrospray ionization (ESI) source ApolloII , as previously described byWillkommen et al.75 In brief, total concentrations of Cu, Fe, Mn, andSe in CSF were quantified using a dynamic reaction cell mass spectrometerNexIon DRC from PerkinElmer . 103Rh (1 μg L–1) was used as an internalstandard for all elements. Ion exchange chromatography using a BeckmanSystem Gold 127NM Solvent Module  high-performance liquid chromatography system, equipped with9725i PEEK injection valve from Rheodyne and degasser Degassex Model D6-4400 was used for the trace element species separation.75 The following columns were used: a Dionex AS-11 ion exchange column(250 × 4 mm i.d.) for Se species and an analytical cationic columnDionex IonPac CS5A RFIC 4*250 mm with a guard column Ion-PacCG5A  for redox species of Fe,Cu, and Mn. Double focusing sector field ICP–MS  was used for the detectionof Fe, Cu, and Mn species at medium resolution mode (m/Δm = 4000) and Se in high-resolution mode.75Quantification of total Se, Fe,Mn, and Cu in CSF and speciation of these elements were performedas reported in previous publications.Supporting Information). To enhancethe comprehensiveness of our analysis, we integrated the data frommetallomics and metabolomics, allowing us to obtain two distinct typesof measurements for each sample. By combining information from bothfields, we gained a more comprehensive understanding of the molecularlandscape associated with ALS.77 The dataset was unitvariance-scaled and analyzed through an OPLS-DA. The Y variables were set to dichotomous values: 0 = control, 1 = ALS.The model was tested for the ability to classify and discriminate.Hotelling’s T2 test (95%) was applied to prohibit the influenceof strong outliers on the models. The classification performance wasevaluated using sevenfold cross-validation. As a result, the modelinferred strongly interrelated masses and metallomics variables, anda list of the most important masses were defined, choosing the highestloadings values. We isolated them in a list of masses (Table S4) that we denominate core metabolome.33 For each variable, we have calculated the p-values and the false discovery rate. Moreover, we addedthe AUC values with the fold change values. Those masses drive themain separation between the two classes. By further studying and evaluatingsuch lists of correlated variables, we found them to show a particularalteration in ALS patients from a biological point of view. Moreover,we focused on the reduced list of biomarkers to find a possible biologicalinterpretation and insight into the metallomics and metabolomics relations.Another statistical model was applied, sparse PLS-DA. This confirmedthe relations between the two datasets and the ability to separatethe case from the control groups. Those elaborations were done inSIMCA 13.0.3.0  and with reportROCand MixOmics packages .Initially, our metabolomics dataset consistedof 7695 MS features. To refine and focus our analysis on metabolitesof interest, we applied a filtering step to exclude features associatedwith the presence of multiple heavy atoms, such as sulfur or phosphorus.This resulted in a reduction of the dataset, narrowing it down to1745 features. For the metallomics measurements, we used the totalconcentrations and the species characterization. Here, all the variablesare reported that were included in the statistical evaluation  and HMDB (https://hmdb.ca/)were used for the identification of the metabolites and metabolitecategorization.For the metabolomics data library,searches were executed using an R script based on the MassTRIX approach.


Portulaca oleracea (PO) is a commonly known medicinal crop that is an important ingredient for traditional Chinese medicine (TCM) due to its use as a vegetable in the diet. PO has been recorded to be frequently adulterated by other related species in the market of herbal plants, distorting the PO plant identity. Thus, identification of the botanical origin of PO is a crucial step before pharmaceutical or functional food application. In this research, a quick assay named “loop-mediated isothermal amplification (LAMP)” was built for the specific and sensitive authentication of PO DNA. On the basis of the divergences in the internal transcribed spacer 2 (ITS2) sequence between PO and its adulterant species, the LAMP primers were designed and verified their specificity, sensitivity, and application for the PO DNA authentication. The detection limit of the LAMP assay for PO DNA identification specifically was 100 fg under isothermal conditions at 63 °C for 30 min. In addition, different heat-processed PO samples can be applied for use in PO authentication in the LAMP assay. These samples of PO were more susceptible to the effect of steaming in authentication by PCR than boiling and drying treatment. Furthermore, commercial PO samples pursued from herbal markets were used to display their applicability of the developed LAMP analysis for PO postharvest authentication, and the investigation found that approximately 68.4% of PO specimens in the marketplace of herbal remedies were adulterated. In summary, the specific, sensitive, and rapid LAMP assay for PO authentication was first successfully developed herein, and its practical application for the inspection of adulteration in PO samples from the herbal market was shown. This LAMP assay created in this study will be useful to authenticate the botanical origin of PO and its commercial products. Portulaca oleracea (PO), generally recognized as purslane, is classified into Portulacaceae and is popularly distributed worldwide for use as a vegetable and medicinal crop. Regarding dietary nutrition, PO contains rich omega-3 polyunsaturated fatty acids and various vitamins and minerals; thus, PO is frequently utilized in mixed salad or broths as a functional potherb [Portulaca species and Bacopa monnieri (BM) demonstrate similar morphological features between POs [ potherb ,2. PO al potherb ,5. As a  potherb ,7. For eween POs . These hween POs ,7.Notably, in the official herbal pharmacopeia of traditional Chinese medicine (TCM), PO is solely authenticated as having the correct botanical origin for use and is habitually applied for cold blood and the treatment of dysentery, as well as infections . Based oTo date, direct analytical approaches such as macroscopic examination, microscopic examination, and thin-layer chromatographic analysis have been applied for PO authentication ,8. HowevIn this work, DNA authentication of PO by a fast and species-specific LAMP assay was developed. Derived from the internal transcribed spacer 2 (ITS2) loci sequence variation between PO and its related adulterants, LAMP primers were designed to evaluate their specificity, sensitivity, and reactivity during DNA amplification for PO authentication. In addition, different heat-treating methods to process PO plants were likewise evaluated by the LAMP assay to assess its applicability for the authentication of PO on the market. Finally, commercial PO samples were practically tested for authentication by this validated LAMP analysis. To the best of our knowledge, this created LAMP assay is the first report demonstrating PO botanical origin identification for authentic application on herbs.Portulaca oleracea (PO) authentication [ITS2 has been recognized as a potential DNA barcode for tication . To builThe sequences of the PO02 LAMP primer set illustrated their respective annealing positions on the ITS2 template DNA of PO, as depicted in To evaluate the detection sensitivity by LAMP, distinct amounts of PO genomic were DNA prepared for use as templates to conduct the reaction. The results exhibited that 10 pg of PO genomic DNA was minimal demanded during the detection by the PO02 LAMP primer set , Lane 4.The heating procedure is popularly applied to process herbal materials for TCM production. However, heat processing frequently results in DNA damage. Regarding practical applications, heat-treated PO plant samples were assembled and tested for PO DNA verification with the LAMP analysis. To imitate the heating procedure, three methods, boiling, steaming, and drying, were applied to process the PO sample. As elucidated in Even in the samples treated with boiling for 30 min, steaming for 30 min, and drying for 7 days, PO DNA was authenticated by the LAMP assay. This manifested that the PO authentication with LAMP was not affected even when it was processed with strict steaming conditions. The assay could still be performed successfully when heat-processed PO was used. In contrast to diagnostic PCR, however, the PO sample, which was steam processed for 30 min, did not exhibit any specific PCR product of 186 bp after the diagnostic PCR assay was performed B. CollecTo examine the validated LAMP assay that is competent in investigating the PO authenticity of PO commercial products in the market, nineteen PO samples and their related products gathered from numerous herbal stores were employed to detect the authenticity by LAMP . Apart fIn recent decades, herbal materials and their demands have become more prevalent and important because complementary and alternative medicine (CAM) has become broadly accepted and utilized in countries around the world ,5. HowevBacopa monnieri (BM), belonging to Plantaginaceae, demonstrated higher sequence variation between PO-related species. The criteria were sufficiently met to further design LAMP primers for PO authentication or the discrimination of adulterants.In this investigation, PO authentication by LAMP using ITS2 as a molecular marker was first successfully developed. The ITS2 sequence variation between PO and PU, PQ, PPS, PPI, and BM ranged from 8% to 30%. These results reflected the great variation of ITS2 regions for LAMP primer designation. Because most of these plants were devised from the same classification of taxonomy, particularly PO, PU, PQ, PPS, and PPI, a lower distant genetic relationship between them and PO was expected. In contrast, the ITS2 sequence of During the LAMP reaction using specifically designed LAMP primers, there is no need to perform the DNA denaturation step by heating to a high temperature. Thus, substantial reaction time for DNA amplification is saved. In contrast, PCR requires repeating cycles of heating and cooling for DNA amplification. Practically, this is the main reason why PCR requires more time than LAMP. For the LAMP reaction, primer design is a critical step in performing DNA amplification. However, the complexity of LAMP primer design is higher than that of PCR, even though the software is currently available for primer design assistance. In this work, three LAMP primer sets, PO01, PO02, and PO03, were designed and selected for the evaluation of LAMP reactivity . SignifiThis established LAMP assay can be used not only to verify the heat-processed PO samples but also for PO authentication on the herbal market . Due to During authentication by LAMP, six of nineteen PO samples were detected, and their PO identities were verified. The diagnostic consequences of PO authentication by LAMP exhibit complete agreement with the microscopic examination . In contPortulaca species, including Portulaca oleracea (PO), Portulaca umbraticola (PU), Portulaca psammotropha (PPS), Portulaca pilosa (PPI), and Portulaca quadrifida (PQ), were collected throughout Taiwan. Bacopa monnieri (BM) plants in Taiwan were also gathered, and all the above-obtained samples were authenticated by Dr. Wen-Te Chang at the LiFu Museum of Chinese Medicine at China Medical University (CMU) based on taxonomy and DNA sequences and were used as standard herbal samples  according to the manufacturer’s procedures. The purified DNA was stocked in a freezer at −20 °C, and the DNA concentration was analyzed using a spectrophotometer at 260 nm.https://www.ncbi.nlm.nih.gov/genbank/, accessed on 26 June 2022), as submitted previously [http://www.ebi.ac.uk/clustalw/). The sequence alignment of ITS2 on the six species is shown in https://primerexplorer.jp/e/) derived from the ITS2 sequence variation between PO and its related adulterants. All designed LAMP primers for the authentication of PO are shown in The ITS2 DNA sequences of all species used in this study were attained from the GenBank of NCBI , 6 mM MgSO4 , 1.4 mM dNTP Mix , 1.6 μM of respective inner FIP and BIP primer, 0.2 μM of respective outer F3 and B3 primer, 8 U of Bst DNA Polymerase , and different amounts of extracted genomic DNA, and the rest was mixed with sterile water to a total volume of 25 μL. After mixing well, the reaction was carried out in a 96-well thermal cycler . The LAMP conditions were implemented at 63 °C for 60 min and then followed by a heating treatment for 10 min at 80 °C for the inactivation of enzyme catalysis. After the reaction, the resultant LAMP products were stocked at 4 °C up to further analysis. Optionally, to determine the effects of loop primers ) to achieve DNA electrophoresis for 25 min, and then the gel was placed on an electrophoresis transilluminator  for observation of the LAMP product. Alternatively, 10 µL of LAMP product was mixed with 1 µL of 1000× diluted SYBRTM safe DNA gel stain , and then the stained LAMP product was observed directly by the naked eye under UV light illumination.After the LAMP reaction, each LAMP product with 5 μL was loaded onto a 2% agarose gel (containing 0.01% SYBR green dye (SYBRPortulaca oleracea (PO) and other specimens  used in this work are revealed in To determine the optimal temperature for performing the LAMP assay, different temperatures of reaction varying from 60 to 68 °C were applied in the LAMP assay under the addition of ten nanograms of extracted PO genomic DNA. After the reaction was performed at different reaction temperatures, the resultant products were examined for the production and DNA intensities of the LAMP products. The optimal temperature used in the LAMP assay was the reaction temperature employed if the highest DNA intensity of the LAMP product was observed.To determine the minimal time of reaction required for performing the LAMP assay, different reaction times, including 10, 20, 30, 40, 50, and 60 min, were used and examined under the addition of ten nanograms of extracted PO genomic DNA as a template. After the LAMP reaction was set up for different reaction times, each produced resultant product was examined to evaluate the production of the LAMP product.To confirm the specificity of the LAMP product, each LAMP product was examined by BstNI  digestion. The mixture for the digestion reaction contained 10× buffer R, 20 U of BstNI, and 2 µL of LAMP product, and the remaining volume was deionized water added to yield an overall volume of 25 μL. The digestion reaction was performed at 37 °C for 4 h. After the reaction, a 5 μL aliquant of the LAMP product was checked through electrophoresis with a 2% agarose gel.To decide the detection sensitivity of LAMP, the PO genomic DNA with different quantities  were prepared and employed as templates for the LAMP assay. After the LAMP analysis was completed, the minimal DNA used in the assay was determined as the detection limit for initiating the LAMP reaction.TM SYBRTM Green Master Mix  and 0.4 µM of each primer. A PCR thermal cycler was employed to perform DNA synthesis using the subsequent cycling conditions: early denaturation at 95 °C for 30 s, DNA denaturation with 35 cycles at 95 °C for 30 s, primer annealing at 58 °C for 10 s, extension of the synthesized DNA at 72 °C for 1 min, and the end of a final extension at 72 °C for 1 min. After the reaction, a 5 μL aliquot of PCR product for each specimen was addressed by electrophoresis with a 2% agarose gel.For the detection of PO DNA from all specimens by species-specific PCR, the onward primer PO02-F (5′-GCTTCGCGTCTCCCCC-3′) and backward primer PO02-R (5′-CCAACAAGCCCATCCCAACA-3′) based on the ITS2 DNA sequence were designed and used for PO authentication. For the 5.8S ribosomal DNA amplification as a reference to all extracted DNA samples, the forward primer 5.8S-F (5′-AACGACTCTCGGCAACGGATA-3′) and reverse primer 5.8S-R (5′-AACTTGCGTTCAAAGACTCG-3′) were used for the synthesis of internal control DNA. The reaction mixture volume used for the PCR was 25 µL, which contained 1× PCR buffer for PowerUpFresh PO plants were harvested and processed with three heating methods: boiling, steaming, and drying. For boiling treatment, the plants were subjected to boiling water for heating for 10, 20, and 30 min; for steaming treatment, the plants were sterilized in a high-pressure autoclave at 1.5 atm at 121 °C for 10, 20, and 30 min. With respect to drying, the plants were baked in an oven at 40 °C for 1, 4, and 7 days. The purified genomic DNA of these heat-processed PO specimens was used as the template for LAMP and PCR assays.In conclusion, the PO DNA identification by LAMP analysis was first evolved in this exploration. Under isothermal circumstances, PO authentication with high specificity, sensitivity, and quickness was performed by specific DNA amplification. The LAMP assay for PO authentication is very useful as a model for establishing a molecular authentication method for on-site application to the identification of other medicinal crops.


This study aimed to summarize the clinical features of Autoimmune Glial Fibrillary Acidic Protein Astrocytosis mimicking tuberculosis meningitis to improve clinicians’ understanding of this disease.We retrospectively analyzed the clinical manifestations, cerebrospinal fluid results, and imaging data of five patients with Autoimmune Glial Fibrillary Acidic Protein Astrocytosis mimicking tuberculous meningitis who were admitted to Xiangya Hospital Central South University between October 2021 and July 2022.Five patients were aged 31–59 years, with a male-to-female ratio of 4:1. Among the cases reviewed, four had a history of prodromal infections manifesting as fever and headache. One patient developed limb weakness and numbness with clinical manifestations of meningitis, meningoencephalitis, encephalomyelitis, or meningomyelitis. Cerebrospinal fluid analysis revealed an increased cell count in five cases, with a lymphocyte majority. All five cases had a CSF protein level > 1.0 g/L, CSF/blood glucose ratio < 0.5, and two patients had CSF glucose < 2.2 mmol/L. Decreased CSF chloride was observed in three cases, while increased ADA was observed in one case. Both serum and cerebrospinal fluid were positive for anti-GFAP antibodies in three cases, while in two cases, only CSF was positive for anti-GFAP antibodies. Additionally, hyponatremia and hypochloremia were observed in three cases. No tumors were detected in any of the five patients during tumor screening, and all five cases had a good prognosis following immunotherapy.Anti-GFAP antibody testing should be routinely performed in patients with suspected tuberculosis meningitis to avoid misdiagnosis. Autoimmune glial fibrillary acidic protein astrocytopathy (A-GFAP-A) is a group of autoimmune neurological diseases , 2 affecThis research retrospectively analyzed five patients with A-GFAP-A and clinical features of tuberculous meningitis who were admitted to the Neurology Department of Xiangya Hospital of Central South University between October 2021 and July 2022. The patients satisfied the following inclusion criteria[The collected and analyzed clinical data included age, gender, first symptoms, main clinical manifestations, laboratory tests, imaging tests, treatment plans, and follow-up prognoses by phone.This study used a cell-based assay (CBA) method because of its high specificity and sensitivity. CBA is a cell transfection-based indirect immunofluorescence assay. In this assay, GFAP genes are transfected into mammalian cells to express GFAP antigens in the mammalian cytoplasm. Green fluorescent proteins (GFP) were also expressed in the transfection as an internal reference for detection. The transfected cells were then fixed onto 96-well microplates to make antigen plates. GFAP antibodies in human serum, plasma, and cerebrospinal fluid samples were detected semi-quantitatively using indirect immunofluorescence.The autoantibodies were detected using the CBA method. Briefly, the serum to be tested was diluted 1:10, or the original fluid of cerebrospinal fluid was used to incubate the patch of cells at room temperature for 60 min. The patch of cells was then treated with detergent three times, for five min each. The FITC-labeled goat anti-human IgG secondary antibody was then diluted 1:50 and incubated in the cell patch for 30 min at room temperature. The patch of cells was then cleansed, sealed, and observed under a fluorescence microscope. The fluorescence signal was significantly higher than the background signal, allowing the serum in the cell membrane to be identified as the positive serum of autoantibodies. When testing the titer of the positive serum, the serum or cerebrospinal fluid was diluted in a gradient, and the method described above was followed. The titer value of the positive serum was determined as the highest dilution multiple of the detectable positive signal.The prognosis was assessed using the modified Rankin Scale (mRS). An mRS score of 0 ~ 2 was considered a good prognosis, whereas a score of > 2 was considered poor.The clinical characteristics are summarized in Table 6/L), predominantly lymphocytes, was observed in five patients. Elevated CSF protein levels with values > 1.0 g/L (reference range: 0.15–0.45 g/L) were observed in all five cases. The CSF/blood glucose ratio < 0.5 was observed in all five cases, and in three cases, the absolute CSF glucose level (reference range: 2.5–4.4 mmol/L) was reduced. All five patients had reduced CSF chloride levels (reference range: 120–130 mmol/L), while one patient had elevated adenosine dehydrogenase (ADA) (reference range: 0–4 U/L). CSF immunoglobulin studies revealed that coxsackie virus IgM and human herpesvirus IgM were positive in one case, respectively. All five patients were negative for CSF mycobacterium tuberculosis smears, CSF mycobacterium tuberculosis cultures, CSF mycobacterium tuberculosis antibodies, CSF bacteriological culture, and CSF fungal culture  findings: Three patients had normal cerebrospinal fluid pressure, while the other two were presented with elevated pressure (reference range: 80–180 mmHg). High white blood cells count  and hypochlorhydria , serum tumor marker, and thyroid function tests did not reveal any abnormalities.The results of MRI findings are displayed in Table Video EEG tests were performed in three cases, one of which revealed diffuse slow waves.In the acute stage, all patients reviewed in this study received either glucocorticoid shock therapy or combination therapy with plasma exchange, and all of them responded well to treatment, as evidenced by the reduced mRS scores. The follow-up period was from the 2nd to the 8th month, with an average follow-up time of four months. During the most recent follow-up, the mRS scores of four cases were less than 2, while the mRS score of the remaining cases was 3, but without recurrence. There was no evidence of tumor recurrence in any reviewed patients during the follow-up period.In this study, all five patients reviewed had a subacute disease, with fever and headache being the first symptoms for four patients. The primary clinical syndromes were meningitis, meningoencephalitis, encephalomyelitis, or meningomyelitis. Pulmonary CT, T-SPOT, PPD skin test, CSF mycobacterium tuberculosis cultures, CSF bacteriological culture, CSF fungal culture, HIV, TPHA, and serum tumor marker were negative for all patients. Their cerebrospinal fluid analysis revealed increased cell counts, with predomination of lymphocytes, elevated CSF proteins > 1.0 g/L, and CSF/blood glucose ratio < 0.5. All cases tested positive for GFAP antibody in CSF, and their response to corticosteroid therapy was excellent, confirming the diagnosis of A-GFAP-A. The clinical symptoms, cranial imaging changes, and other CSF anomalies of the five A-GFAP-A patients reported in this study were similar to those of tuberculous meningitis (TBM). According to previous studies, the misdiagnosis rate ranges between 4.5% and 35.7% [In this study, all five patients underwent brain MRI scans enhancement and demonstrated no enhancement. The brain MRI images of two patients depicted abnormal T2-weighted and FLAIR imaging of hyperintensity signals in the bilateral basal ganglia. Moreover, the brain MRI images of one patient exhibited abnormal T2-weighted and FLAIR imaging of hyperintensity signals in the bilateral thalamus lesion. Although previous studies indicate that linear perivascular radial gadolinium enhancement in the brain is a characteristic manifestation of the disease , 10, 11,In this study, the similarities and differences between A-GFAP-A and TBM were primarily discussed regarding cerebrospinal fluid changes to improve the understanding of clinicians about the disease. In this study, five cases had elevated CSF cell counts, with lymphocytes predominating. Elevated CSF proteins > 1.0 g/L were also observed in all five cases. Elevated CSF lymphocytes, protein is more common in TBM; autoimmune disease is uncommon. Studies have revealed that the cerebrospinal fluid of A-GFAP-A patients is predominantly characterized by inflammatory changes and that CSF lymphocytes and proteins can be increased, with some patients exhibiting CSF protein > 1.0 g/L –6, 8–10.In this study, the CSF/ blood glucose ratio in all five cases was < 0.5, and two patients had CSF glucose < 2.2 mmol/L. The patient demonstrated decreased CSF glucose but normal serum glucose, which is hardly seen in autoimmune diseases. Studies have shown that the decrease in CSF glucose in A-GFP-A patients is only 15–18% . ReducedIn this study, one patient experienced a transient increase in CSF adenosine deaminase (ADA) activity, which recovered spontaneously. Kimura et al.  reportedWe reported five cases of A-GFAP-A with a clinical presentation and CSF profile resembling TB meningitis. We consider A-GFAP-A an important differential diagnosis, particularly in TB-endemic regions. In clinical practice, CSF GFAP-igg should be considered for early evaluation in patients with suspected tuberculous meningoencephalitis and atypical characteristics, and diagnostic antituberculosis should be administered with caution.


At baseline, local activity, as quantified by fractional amplitude of low-frequency fluctuations, differentiated between normal and abnormal CSF groups in the NbM but not EC. Further, NbM activity linearly decreased as a function of CSF ratio, resembling the disease status. Finally, NbM activity predicted the annual percentage signal change in EC, but not the reverse, independent from CSF ratio. Our findings give novel insights into the pathogenesis of Alzheimer’s disease by showing that local activity in NbM is affected by proteinopathology and predicts functional degeneration within the EC.Recent models of Alzheimer’s disease suggest the nucleus basalis of Meynert (NbM) as an early origin of structural degeneration followed by the entorhinal cortex (EC). However, the functional properties of NbM and EC regarding amyloid-β and hyperphosphorylated tau remain unclear. We analysed resting-state functional fMRI data with CSF assays from the Alzheimer’s Disease Neuroimaging Initiative  differentiates between normal and abnormal CSF and decreases linearly with CSF ratio. NbM activity predicts signal changes in the entorhinal cortex, but not vice versa, independent of CSF ratio, offering novel insights into Alzheimer’s disease pathogenesis.Mieling   To this end, we used data from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and performed a longitudinal region of interest (ROI) analysis over 2 years, focusing on regional and interregional resting-state functional MRI (rsfMRI) properties. In detail, we analysed (i) the fractional amplitude of low-frequency fluctuations (fALFF) to quantify spontaneous neuronal activity,10-12 (ii) regional homogeneity (ReHo) reflecting the synchronicity of neural activity between a voxel and its neighbouring voxels13 and finally (iii) the functional connectivity between NbM and EC. While all three measures may help to gain new insights into Alzheimer’s disease progression, we initially focused on fALFF given its established role14-16 and report ReHo and functional connectivity analyses in the Since functional brain changes in Alzheimer’s disease often precede structural degeneration,In the first step, baseline signals and longitudinal functional changes were compared based on harmonized CSF assays of Aβ and pTau in NbM and EC. Subsequently, we investigated functional changes in disease progression using the CSF markers. Finally, we tested the competing models NbM→EC versus EC→NbM on a functional level. Our main hypothesis was that functional signals in the NbM predict functional changes in EC, which would provide further evidence supporting the pathological staging model from NbM to EC. From a more general perspective, we aimed to provide new insights into the underlying functional properties of Alzheimer’s disease, which may contribute to further developing markers and treatment strategies.Data used in the preparation of this article were obtained from the ADNI database (adni.loni.usc.edu). ADNI was launched in 2003 as a public–private partnership, led by Principal Investigator Michael W. Weiner, MD, to test whether serial MRI, PET, other biological markers and clinical and neuropsychological assessment can be combined to characterize the progression of mild cognitive impairment (MCI) and early Alzheimer’s disease.https://ida.loni.usc.edu). Specifically, we only selected data from participants with CSF biomarkers and two rsfMRI scans acquired with a delay of 2 years with the same MR scanner and head coil to ensure within-subject comparability.Since rsfMRI was not acquired in all ADNI cohorts, here, data were combined from ADNI-GO, ADNI-2 (ADNI-GO/2) and ADNI 3, downloaded from the Image and Data Archive (IDA) platform run by the Laboratory of Neuro Imaging (LONI) . Further, we only included MRI data with excellent, good or fair quality. For further information on image acquisition, see the http://adni.loni.usc.edu.Participants were scanned at multiple sites equipped with 3-Tesla MRI scanners according to unified ADNI monitoring protocols.http://rfmri.org/dpabi) toolbox version 5 (release 5.2_210501), which is based on the Statistical Parametric Mapping toolbox  for MATLAB®. It started with the removal of the first 10 volumes and subsequently included the following steps (i) slice time correction; (ii) spatial realignment; (iii) T1 co-registration to the mean functional image; (iv) CSF, grey and white matter tissue segmentation and spatial normalization using diffeomorphic anatomical registration using exponential lie algebra (DARTEL)18 for T1 images; (v) regression of nuisance variables; and (vi) normalization to the Montreal Neurological Institute (MNI) space and resampling to an isotropic voxel size of 3 mm of the functional images using the parameters estimated by DARTEL  by overlaying a ROI mask in standardized space; especially, the EC represents a region that might often be affected by artefacts.21 To better understand how both relate to functional degeneration in NbM and EC, we followed previous studies2 and used ADNI’s CSF samples, produced with a fully automated Elecsys® protocol of Aβ and pTau from the first measurement (t1). For each participant, we extracted Aβ 1–42 and pTau181 values. Since the protocols by Elecsys® are still under development, the results are restricted to a specific technical limit (>1700 pg/mL). Higher values were provided by extrapolation of the calibration curve for research purposes only but not diagnostics. Further information on CSF draws and analyses can be found at http://adni.loni.usc.edu.Alzheimer’s disease neuropathology includes the accumulation of Aβ resulting in plaques and pTau leading to neurofibrillary tangles.23 Based on these findings, the standardized and cross-validated cut-off of 0.028 was used to divide the participants into an abnormal (pTau/Aβ ≥ 0.028) and a normal (pTau/Aβ < 0.028) CSF group.23 Importantly, no participant classified with Alzheimer’s disease had a normal CSF ratio, but a few (n = 10) participants classified with MCI did, which indicates an unclear aetiology. Nevertheless, we included them based on biological instead of a syndromal grouping.24Here, we analysed both proteins by using a previously established ratio of pTau/Aβ, which is known to highly concord with PET measures and clinical diagnoses.t1 scores are used, including validated memory (MEM) and executive function (EF), based on a confirmatory factor analysis.26 Memory scores include the Alzheimer’s Disease Assessment Scale, Logical Memory test, Mini-Mental State Examination (MMSE) and Rey Auditory Verbal Learning Test (RAVLT). EF scores are based on the Category Fluency, Digit Span Backwards, Digit Symbol Substitution, Trails A and B and the Clock Drawing tests.26 We were also interested in the Montreal Cognitive Assessment (MoCA), Sum of Boxes in the Clinical Dementia Rating Scale (CDRSB) and the Alzheimer’s Disease Assessment Scale-Cognition Subscale, 13 tasks (ADAS-Cog 13) to get a deeper understanding of the participants’ cognitive profiles (see below).All participants underwent a comprehensive neuropsychological test battery. Here, t1 diagnosis made by the ADNI Clinical Core: cognitive normal (CN) , MCI  and Alzheimer’s disease . These classifications represent widely used cognitive and functional measures in clinical trials.27-29 Further information regarding diagnostic is available at http://adni.loni.usc.edu.Furthermore, we included participants’ t1 measurement in the analyses. To maximize the number of subjects, the second measurement was selected after an interval of 1.5 years ±12 months (t2).1 Further details on inclusion and exclusion criteria for participating in ADNI are available under http://adni.loni.usc.edu. In total, 153 participants for ADNI-GO/2 and 141 for ADNI 3 (only basic rsfMRI version) fulfilled our inclusion criteria. However, a large proportion had to be excluded mainly based on fMRI data quality  and abnormal CSF  values  temporally related to a rsfMRI acquisition (e.g. a participant’s screening MRI and baseline lumbar punction measurement). This measurement served as lity see . Thus, d) values .t1).http://adni.loni.usc.edu). The analyses presented here were approved by the local Ethics Committee of the University of Lübeck and carried out after ADNI’s recommendations including the approval of the manuscript before submitting to a journal.Each centre collecting data for ADNI provided an Institutional Review Board (IRB) approval and meets ADNI’s requirements. Informed consent was obtained from all ADNI participants  to compare baseline signals and the annual percentage signal change  between regions (NbM and EC as a within-subject factor) and CSF groups . Covariates such as age, sex, education, ADNI cohort and scanner manufacturer were included to adjust for different scan protocols and other potential scanner-related differences. All 2 × 2 (region × CSF group) mixed ANCOVAs were carried out in IBM SPSS statistics version 25 (SPSS) with type III sums of squares, and within-subject effects were interpreted without covariates.Z-scored data. Subsequently, the dependent overlapping correlations of NbM versus EC with CSF ratio were compared using cocor.32To better understand the relationship between disease status and functional MRI properties, CSF ratios (see section CSF biomarker) and functional MRI signals were considered in a linear regression model in SPSS. The functional MRI signal served as dependent variable and CSF ratio as independent variable. The regression was run with the ® R2020b with fitlm using the bisquare weight function with the default tuning constant. The same covariates as for the mixed ANCOVA were included in the model. Finally, the predictive models (NbM→EC and EC→NbM) were tested for each CSF group  and each functional property (fALFF and ReHo). The data were Z-scored before entering the analysis to ensure comparability of the APSC and baseline signal.To minimize the influence of outliers, especially in the APSC, robust regression models were carried out in MATLAB33 for fALFF and ReHo investigating whether CSF group assignment moderates the spread (NbM→EC versus EC→NbM) of functional degeneration. Here, CSF group was used as a dichotomous moderator variable. For the construction of products mean-centering was applied, and the heteroscedasticity consistent standard error HC3 (Davidson–MacKinnon) was applied.Moderation analyses were carried out in SPSS using the PROCESS macro34 was used to assess longitudinal APSC in fALFF and ReHo. It accounts for the days between both measurements and minimizes the influence of differences between both measurements within a subject.The following formulaBased on the CSF grouping strategy, we investigated how abnormal CSF and normal CSF groups performed in neuropsychological tests. For each test, one-way fixed effect ANOVAs were carried out with CSF group as factor and age, sex and education as covariates. As expected, the normal CSF group is less affected by cognitive impairment than the abnormal CSF group see .F = 7.943, P = 0.006, η2p = 0.112; F = 4.623, P = 0.035, η2p = 0.068; t(69) = 3.141, P = 0.002) but not EC (t(69) = 1.856, P = 0.068]. There was no main effect of region  and no significant group × region interaction  in APSC fALFF  = 9.437, P = 0.003; R²=0.031, F = 2.206, P = 0.142; Z = −1.94; P = 0.0262; 95% CI: −0.3429 to 0.0015). Additionally, we analysed both linear regressions independently for Aβ and pTau  = 4.19, P = 0.044; R²=0.09, F = 6.784, P = 0.011; R²=0.022, F = 1.556, P = 0.217; R²=0.003, F = 0.234, P = 0.63; In a next step, we used linear regressions on baseline fALFF values from NbM and EC, respectively, with CSF ratio as independent variable. It revealed a significant linear effect in the NbM . The other regression model EC→NbM did not show a significant effect , with a significant direct effect of NbM→EC , but no significant moderator effect , which is in line with the robust regression analysis.Finally, we performed two moderation analyses. The first model included baseline fALFF NbM as independent variable, fALFF EC APSC as dependent variable and CSF group as moderator. The model was statistically significant , which, again, is in line with the robust regression analysis.The second model included baseline fALFF EC as independent variable, fALFF NbM APSC as dependent variable and CSF group as moderator. The model was not statistically significant [The results for ReHo  and funcWe investigated the functional properties of the human NbM and EC in relation to the disease progression of Alzheimer’s disease based on longitudinal rsfMRI data and CSF markers of Aβ and pTau. With a focus on fALFF, our data provide evidence that spontaneous local brain activity in the NbM, but not EC, is reduced with CSF ratio, and, importantly, it predicts the APSC in the interconnected EC independently from proteinopathology. As such, our findings extend previous anatomical studies in humans and animals by providing novel physiological insights into the pathological staging model of Alzheimer’s disease suggesting the NbM as an early origin for subsequently affected brain regions possibly via a trans-synaptic mechanism.35 and the NbM is particularly vulnerable to the early accumulation of pTau36-38 and Aβ deposition.39 This may be due to the fact that cholinergic basal forebrain neurons have rather large axons and arbors reaching into the entire central nervous system with high metabolic demands for maintenance, reparation and transportation.40 At the same time, simply due to their sizes, they are more vulnerable to toxins,41 which may further promote disease progression.Local spontaneous brain activity, as quantified by fALFF, was reduced in the NbM at baseline in the abnormal CSF group , and the1 Our functional data support such a view by showing that the NbM’s baseline fALFF signal predicted the APSC in the EC  framework, which includes markers of Aβ, tau (T) and neurodegeneration (N).24 However, this requires a much larger sample of subjects than n = 71, since at least three groups would need to be included , and their distribution is typically unbalanced . Furthermore, the necessary cut-off values, which are often based on PET but in some studies CSF, are not well defined.24 Finally, we wanted to analyse our data as similar as possible to Fernández-Cabello et al. (2020) in order to be comparable.Finally, ADNI is a large multicentre study offering a rich and unique data set. This also means that our rsfMRI data come from different MR scanners, possibly leading to a bias in image quality and extracted signal. Therefore, we only included high-quality data based on comparable protocols and within-subject measurements from the same scanner. We also employed appropriate covariates in our statistical models, and differences in scanning parameters (e.g. slice order or number of volumes) were accounted for during preprocessing.Functional activity in the human basal forebrain scaled with proteinopathology and predicted the functional decline within the interconnected EC independent from CSF status. As such, our findings extend the pathological staging model of Alzheimer’s disease by giving novel insights into the functional properties of the underlying brain regions. From a more general perspective, fALFF appears to be a suitable marker to further investigate functional brain changes associated with the progression of Alzheimer’s disease.fcad262_Supplementary_DataClick here for additional data file.


Liver metastasis is one of the most important factors affecting the prognosis of patients with neuroendocrine neoplasms (NENs). Transhepatic artery embolization (TAE) is the main local treatment of NENs with liver metastasis (NENLM). This study aimed to elucidate the differences between pancreatic and rectal NENLM with a discrepancy in blood supply.A total of 32 patients with NENLM of different primary sites received 102 TAE treatments at our hospital. Clinical features, such as age, sex, World Health Organization (WHO) tumour grade and progression‐free survival (PFS), were compared between patients with pancreatic and rectal NENLM with different blood supplies. The total follow‐up time is 1–5 years.p = 0.005). Tumour shrinkage in all cases with NENLM was 50% with an objective response rate of 37.5%, disease control rate of 75% and PFS of 12 months. Disease progression (p = 0.09), tumour shrinkage (p = 0.07) and death (p = 0.19) were more prominent in the pancreatic NENLM group than in the rectal NENLM group. Progression‐free survival was not reached in the pancreatic NENLM group, which was more prominent than in the rectal NENLM group . The main adverse events were abdominal pain (71.9%) and transaminase elevation (50%), which were more common in pancreatic NENLM than in rectal NENLM.There were 12 cases with tumours originating from the rectum or pancreas, respectively. Other tumour‐originated sites included the duodenum , the thymus and lung , and the unknown . The average age of patients was 51.59 years, and 17 (53.1%) were men. WHO grade 1, 2 or 3 tumours occurred in three (9.4%), 23 (71.9%) and six (18.7%) patients, respectively. Hepatic tumour burdens of low (<25%), middle (25%–50%) and high (>50%) levels were found in 13 (40.6%), eight (25%) and 11 (34.4%) patients, respectively. There were more patients with hypervascular pancreatic NENLM than with hypovascular rectal NENLM (Transhepatic artery embolization treatment is markedly effective and safe for treating NENLM, especially pancreatic NENLM. According to the Surveillance, Epidemiology, and End Results database, the incidence of NENs has shown a significant upward trend in recent years, increasing 6.4 times to 6.98/100,000 from 1973 to 2012. The gastrointestinal tract and pancreas are the most common primary sites of NENs, accounting for nearly 60% of all cases.According to the tumour proliferation index (Ki‐67) and mitotic data in WHO 2019, NENs are divided into the well‐differentiated neuroendocrine tumour (NET) grades G1, G2, G3 and poorly differentiated neuroendocrine carcinoma.Transcatheter arterial embolization (TAE) can block the blood supply of lesions and shrink tumours in hypervascularized primary and metastatic liver tumours, such as hepatocellular carcinoma.In this study, we investigated the efficacy and safety of TAE therapy for 102 times in 32 patients with NENLM at our hospital. We found that TAE significantly improved PFS for those patients, exhibiting better outcomes when used on pancreatic than rectal NENLM.22.1This cohort study was approved by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University. The patient and family members signed an informed consent form for TAE. Patients with NENLM who received TAE in our hospital from January 2018 to March 2021 were included. We followed up with the patients by telephone for 1–5 years. They had nonfunctional NENs.Inclusion criteria were as follows: (1) NENs and NENLM were confirmed by pathological and immunohistochemical examinations; (2) measurable lesions in type III NENLM without indication for surgery discussed by our multidisciplinary team; (3) performance status score of 0–1 with Child–Pugh grade A; (4) NENLM did not invade large blood vessels; (5) contrast‐enhanced computer tomography (CT) or magnetic resonance imaging (MRI) was completed before and after TAE.Exclusion criteria were as follows: (1) clinical data were not complete, such as missed imaging data or other important therapy; (2) transcatheter arterial chemotherapy embolization (TACE), ablation and other local liver treatments had been performed before TAE except for surgery; (3) contraindication to TAE, such as severe heart, lung, liver or kidney dysfunction.2.2Clinical data included age, sex, primary tumour site, grade according to WHO, surgery history of NENLM and other systemic therapies, such as long‐acting octreotide. For evaluation of blood supply, if the density of the NENLM was lower than that of the surrounding normal liver parenchyma in the arterial phase of contrast‐enhanced CT/MRI, it could be considered a hypovascular type.2.3Tumour shrinkage, objective response rate (ORR), disease control rate (DCR) and progression‐free survival (PFS) were calculated to evaluate the effect of TAE on NENLM and to compare subgroups according to the Response Evaluation Criteria in Solid Tumours RECIST 1.1). The primary endpoints were ORR, DCR and PFS. Efficacy evaluations are divided into complete response (CR), partial response (PR), stable disease (SD) and disease progression (PD). Objective response rate was defined as the rate of CR plus PR. Disease control rate was defined as the rate of CR, PR and SD. Progression‐free survival was defined as the time from TAE to disease progression. The patients underwent enhanced CT or MRI examinations within 7 days before TAE as baseline data. The same imaging examination was performed within 1–3 months after TAE; subsequently, it was followed up every 3 months  were recorded according to the Common Terminology Criteria for Adverse Events version 5.0 (CTCAE v5.0) of the National Cancer Institute.2.4According to the guidelines, systemic therapy of NENs was continued. A total of 24 patients received long‐acting octreotide 30 mg intramuscular‐injection every 4 weeks, and the remaining patients received oral capecitabine and temozolomide for chemotherapy or everolimus 5–10 mg every day for targeted therapy.For TAE, we performed digital subtraction angiography (DSA) to assess tumour blood supply and tumour staining. A 3F microcatheter was catheterized into the tumour supply artery. Superselective embospheres of 40–120 μm diameter  were inserted following the microcatheter according to guidelines. The endpoint of embolization was the stagnation of the secondary branch of the hepatic artery in the area of blood supply of NENLM (the contrast medium was not clear for five cardiac cycles). All patients were treated with short‐acting somatostatin analogs from 1 h before TAE to 48 h after TAE.2.5t‐test or the rank‐sum test. Qualitative data were described by numbers and percentages and compared using the χ2 or Fisher's exact test. Univariate analysis was performed to identify factors associated with PFS. A Cox proportional hazards regression model was used to identify independent prognostic factors associated with PFS. Survival analysis was carried out by the Kaplan–Meier method. Events were defined by disease progression. All statistical significance was defined as p < 0.05.Statistical analysis was performed using SPSS Statistics version 22. Quantitative data are described as the mean ± standard deviation or median (interquartile range) and were compared using the 33.1Thirty‐two patients receiving 102 TAE treatments were included in our study, with an average of 3.19 times per case. Primary sites were the rectum , pancreas , duodenum , thymus and lung  and the unknown . The average age was 51.59, and 17 (53.1%) were men. There were three (9.4%), 23 (71.9%) and six (18.7%) patients with G1, G2 and G3 tumours, respectively, with a median Ki‐67  of 10 . Three patients (9.4%) received NENLM surgery before TAE. Eight (25%) patients were hypervascular of NENLM, and 24 (75%) cases were hypovascular. Low (<25%), middle (25–50%) and high (>50%) hepatic tumour burdens were found in 13 (40.6%), eight (25%) and 11 (34.4%) patients, respectively.p = 0.005). Other characteristics, including age (p = 0.06), sex (p = 0.22), Ki‐67 (p = 0.19), grade (p = 0.67), liver surgery history (p = 0.59), tumour burden (p = 0.83) and long‐acting octreotide therapy (p = 0.5), did not show significant differences between the pancreatic NENLM and rectal NENLM groups , while the DCR was 75% in the pancreatic NENLM group and 66.7% in the rectal NENLM group (p = 0.5). In the pancreatic NENLM group, the risks of PD (p = 0.09), tumour shrinkage (p = 0.07), and death (p = 0.19) were better than in the rectal NENLM group. Five patients died in the rectal NENLM group, while two died in the pancreatic NENLM group  who achieved PR, 12 patients (37.5%) with SD, eight patients (25%) with PD and 16 patients (50%) with tumour shrinkage. The PFS was 12 months Figure , with anp = 0.016; Figure Progression‐free survival was not reached in the pancreatic NENLM group, which was significantly better than in the rectal NENLM group , 0.07–0.76; We cited two patients with TAE treatment as examples. The first was rectal NENLM type III with hypovascular evaluation by contrast‐enhanced CT and DSA , followed by liver damage (50%), vomiting (28.1%) and fever (28.1%). Grade 3 or 4 AEs were abdominal pain (6.3%) and elevation of alanine aminotransferase (6.3%), but two patients developed thrombocytopenia, and one patient developed a liver abscess that prolonged the hospitalization period. We need to pay special attention to the higher incidence of grade 3/4 AEs in the pancreatic NENLM group (41.7%) than in the rectal NENLM group (16.7%). All patients with AEs improved and were discharged after symptomatic treatment. There were no significant differences in the incidence of adverse reactions between the pancreatic NENLM group and the rectal NENLM group . It should be noted that PD occurred in one and two patients in the pancreatic and rectal NENLM group, with two and five patients having died, respectively, which indicated a higher risk of progression and death in the rectal NENLM group.Some reports on the effect of interventional embolization in NENLM exist, but most are based on TACE and transarterial radioembolization (TARE).The incidence of AEs was similar to that in previous studies.Drug treatment remains the main treatment for hypovascular NENLM, and the effect of local treatment has yet to be affirmed. However, NENLM usually has large lesions and tumour load, which seriously inhibits the effect of drug treatment and leads to a poor prognosis for patients. Therefore, we designed this study to observe whether TAE is effective in the local treatment of hypovascular NENLM. In our study, the treatment with TAE of NENLM is safe and effective, even in hypovascular NENLM. Since NENs are relatively rare, the sample size of this study is small. In future, large‐scale clinical studies are needed to explore this issue, and longer follow‐up periods are needed to support the findings of this study.Jian'an Bai: Conceptualization (supporting); investigation (lead); methodology (supporting); resources (supporting); validation (supporting); writing – original draft (lead). Jinhua Song: Data curation (supporting); formal analysis (supporting); funding acquisition (supporting); investigation (lead); methodology (lead); resources (supporting); validation (supporting). Yang Zhang: Data curation (supporting); investigation (supporting); project administration (supporting); resources (supporting); software (supporting); writing – original draft (supporting). Xiaolin Li: Conceptualization (supporting); resources (supporting); validation (supporting); writing – review and editing (supporting). Lijun Yan: Data curation (supporting); investigation (supporting); resources (supporting); software (supporting). Ping Hu: Data curation (supporting); formal analysis (supporting); investigation (supporting); methodology (supporting). Qiyun Tang: Conceptualization (lead); data curation (lead); funding acquisition (lead); supervision (lead); writing – review and editing (lead).There is no conflict of interest to declare.This cohort study was approved by the Ethics Committee of the First Affiliated Hospital with Nanjing Medical University. The patient and family members signed an informed consent form for TAE. All procedures followed the Declaration of Helsinki for research involving human subjects.