Output spike bursts are thought to implement key dendritic computations, such as coincidence detection of bottom-up inputs  and top-down inputs . In this study we used a detailed nonlinear model of L5PC receiving excitatory and inhibitory synaptic inputs to explore the conditions for generating bursts and for modulating their properties. We established the excitatory input conditions on the basal versus the apical tree that favor burst and show that there are two distinct types of bursts. Bursts consisting of 3 or more spikes firing at < 200 Hz, which are generated by stronger excitatory input to the basal versus the apical tree, and bursts of ~2-spikes at ~250 Hz, generated by prominent apical tuft excitation. Localized and well-timed dendritic inhibition on the apical tree differentially modulates Na+, Ca2+, and NMDA spikes and, consequently, finely controls the burst output. Finally, we explored the implications of different burst classes and respective dendritic inhibition for regulating synaptic plasticity.The output of neocortical layer 5 pyramidal cells (L5PCs) is expressed by a train of single spikes with intermittent bursts of multiple spikes at high frequencies. The bursts are the result of nonlinear dendritic properties, including Na The output of most neurons consists of stereotypical electrical pulses, or spikes. Some neurons generate sparse spikes intertwined with groups of spikes in close succession, called bursts. Previous research has implicated bursts in long-range transmission, dendritic computation, and plasticity. However, a detailed account of the spatiotemporal synaptic activation patterns that generate bursts, even in the principal cortical pyramidal neurons, is largely missing. Using experimentally constrained computational modelling we determined the conditions of synaptic activation patterns that induce burst firing in these cells. We found two distinct classes of bursts. They are distinguished by the number of active synapses, on either basal or apical part of the dendritic tree, required for burst initiation, and by the number and frequency of output spikes. We provide an analysis of how location and timing of dendritic inhibition finely edit somatic bursts and show the change in the strength of excitatory synapses during bursts following their dendritic inhibition. Overall, this work offers a deeper understanding of the origin of bursts, including a novel distinction between burst classes and characterization of how inhibition may shape bursts and their correlated synaptic plasticity. Layer 5 pyramidal cells (L5PCs) are considered to be pivotal building blocks of the mammalian neocortex . In rodein vivo tend to fire spikes in a random, Poisson manner such that the timing of each action potential is independent of its predecessors i, values result in “protected” (unchanged) efficacy of the excitatory synapses  to fit experimental findings. Using our model, we tested how L5PC excitatory synapses act under this plasticity rule, and explicitly how inhibition of dendritic Na+, Ca2+ and NMDA spikes (determined by location and Δtinh) controls the manifestation of this plasticity rule.Recent studies associate inhibition of bursts and of dendritic Calearning –44. To sty model ,46  with trunk (1), intermediate tuft (2) or distal (3) inhibition. Note that this maximum value of [Ca2+]i does not fully represent whether an excitatory synapse at the location will be potentiated or depressed, as the time that [Ca2+]i lasts at any given concentration will eventually determine the sign of plasticity. When [Ca2+]i rises from baseline to LTP levels, it goes through LTD levels, so the time above the respective thresholds (together with the learning rate) determines the final synaptic weight (the plasticity sign and magnitude). I.e., if [Ca2+]i crosses θd for a long duration but briefly reaches a maximum over θp, then 2+ hotspot (at location #1) on the apical trunk. The maximum [Ca2+]i at the hotspot is high and nearly invariant to inhibition timing at this inhibitory location  for a magnified effect on synaptic weights. For this proximal inhibition at location #1 the somatic burst is unaffected and therefore so is the Ca+2 spike at the hotspot (blue trace) and the distal NMDA spike (purple trace).ed there C1. Fig 62+ hotspot (at location #2). Well-timed inhibition (|dtinh|< 17 ms) at this location suppressed dendritic spikes, allowing Ca2+ influx only into the distal tuft  do synapses undergo LTP  values for plasticity. In p,d  inhibition of local NMDA spikes controls branch-specific plasticity by transforming LTP to local LTD or to the protected regime, and (3) following the association of bursts, Ca2+ spikes and inhibition with plasticity (see Discussion), we find a correlation between inhibition of bursts and limiting plasticity. The last point is in line with recent experimental studies, showing that limiting plasticity to specific connections relies on inhibition of bursts [We briefly summarize the key results of this section by noting that (1) inhibition located at the hotspot or immediately distal to it suppressed the Caf bursts –44.2+-dependent plasticity rule with whole-tuft excitation as in 2+]i outside the hotspot, meaning minor plasticity effects beyond LTP at the hotspot in the burst-suppressed dendritic tuft .We showed that somatic bursts arise either from a bidirectional stream of excitatory inputs combining basal and tuft synapses, or by generation of a local dendritic voltage plateau due to clustered excitatory input to the apical tuft alone Figs  and S1. ses Figs  and S3. ses Figs , high ex2+-dependent LTP in spines, resembling the classical asymmetric spike-timing dependent plasticity rules (STDP) i (concentrations) during the simulation at different inhibitory locations and times , which creates a more variable and realistic range of phenomena (LTP/D & protected). To generate synaptic weight changes in 2+]i measurements are difficult to control for in these relatively short simulations. Synapses on trees represent predicted weights after learning by this rule.Graphs are created for maximal i measured as in p/d values. The heatmap represents synaptic weight after plastic modification, normalized to initial value and colored on a log2 scale . Modified excitatory synapse location is ordered on x-axis. LTP threshold θp on y-axis. LTD threshold θd is kept at a fixed ratio of 0.6 to θp.[Ca(TIF)Click here for additional data file.S5 Figa. Model neuron as in 2+]i measurement as in 2+ hotspot. b1-3. Maximal [Ca2+]i along the apical dendrite at the three locations (colored electrodes in b) as a function of Δtinh between excitation and inhibition for each inhibition location. Dashed lines mark thresholds, shadings the resulting change–LTP (red), LTD (blue). c1-3. Synaptic weights after repeated execution of the learning rule (Eq (Methods) for 10 input repetitions (60 seconds) and Δtinh = -5 ms at each inhibition distance # . Twenty representative excitatory tuft synapses are color plotted by their respective weights: yellow—LTP (in 2), blue—LTD, and grey—protected.(TIF)Click here for additional data file.


This study aimed to investigate immune-related pathophysiology of the temporomandibular joint (TMJ) osteoarthritis (OA) in young females by analyzing transcriptional profiles of peripheral blood mononuclear cells. The RNA-sequencing (RNA-seq) was conducted on 24 young females with TMJ OA (mean age 19.3 ± 3.1 years) (RNAOA) and 11 age and sex matched healthy controls (mean age 20.5 ± 3.7 years) (CON). RNA-seq datasets were analyzed to identify genes, pathways, and regulatory networks of those which were involved in the development of TMJ OA. RNA-seq data analysis revealed 41 differentially expressed genes (DEGs) between RNAOA and CON. A total of 16 gene ontology (GO) terms including three molecular and 13 biological terms were annotated via the GO function of molecular function and biological process. Through ingenuity pathway analysis (IPA), 21 annotated categories of diseases and functions were identified. There were six hub genes which showed significant results in both GO enrichment analysis and IPA, namely HLA-C, HLA-F, CXCL8, IL11RA, IL13RA1, and FCGR3B. The young females with TMJ OA showed alterations of the genes related to immune function in the blood and some of changes may reflect inflammation, autoimmunity, and abnormal T cell functions. The epidemiology of OA of the temporomandibular joint (TMJ) is different from that of OA of the hand, knee, or hip joint, the incidence of which is associated with aging. Previous reports have already described female preponderance and early onset of the disease, especially from the pubertal phases to the age of early 20 s3. The pathophysiology of the TMJ OA is multifactorial and complex, which includes diverse etiological factors such as prolonged parafunctional habits, abnormal occlusal relationship, sustained masticatory muscle tension, and hormonal imbalance5. Given the early onset of the condition, TMJ OA cannot be considered to be just a simple degenerative disease such as the OA of other joints associated with the aging process, so other etiological factors could be assumed. Nevertheless, the clear pathophysiology of the TMJ OA, particularly in young patients has not yet been elucidated so far.Osteoarthritis (OA) is characterized by degradation of components of the extracellular matrix within the articular cartilage and simultaneous remodeling of the underlying subchondral bone, in association with low inflammatory changes13. These processes involved immune-modulating agents, in both innate and adaptive compartments such as cytokines, chemokines, T cells, and B cells13. TMJ OA has been considered to be a low inflammatory arthritic condition and mainly depends on inflammation and elevated levels of inflammatory mediators and cytokines in the TMJ synovial fluid17. Even though one report suggested the possibility of involvement of systemic immune dysfunction in occurrence of TMJ OA18, the influences of systemic immune function and composition of immune cells in the peripheral blood on incidence and progression of TMJ OA have not been fully clarified.Many emerging evidences have demonstrated the role of immune modulation mechanisms in the development and progression of OA19. However, to the best of the knowledge, there are yet no studies which adopted RNA-seq technology to patients with TMJ OA. Therefore, the molecular and genetic background of TMJ OA remains obscure, and the clinical treatment effects for TMJ OA are limited. Understanding comprehensive molecular profiling of TMJ OA is an essential step in discovering new candidate target molecules that are potentially involved in the pathogenesis of TMJ OA. Hence, the aim of the present study was to investigate the role of the immune-related pathophysiology of TMJ OA by analyzing the transcriptional profiles of RNA from peripheral blood mononuclear cells (PBMC), which identify the differentially expressed genes (DEGs) of young females with TMJ OA.The RNA sequencing (RNA-seq) technology has been utilized as a powerful tool to discover potential molecular mechanisms or therapeutic targets in various diseases21. All female participants in the RNAOA showed erosive osseous bony changes on computed tomography (CT) images indicating the area of discontinuation of the cortical lining and adjacent bone  were enrolled. Twenty-four female patients  with TMJ OA on at least one side of the condyle were consequently recruited from those who attended the TMD and Orofacial Pain Clinic at a university hospital from January, 2019 to November, 2019 (RNAOA). Eleven young females  without any sign of TMD and/or TMJ OA who voluntarily participated in the study served as control (CON). Patients with the following conditions were excluded: history of head and neck trauma prior to at least 6 months prior to study entry; autoimmune diseases, which could affect the systemic levels of inflammation including juvenile idiopathic arthritis, lupus erythematosus, ankylosing spondylitis; craniofacial anomalies; and neurodegenerative disorders. All participants in the RNAOA did not show any sign of capsule or myofascial pain in the orofacial area for at least 3 months before enrollment and had not been suffered from chronic pain which last for more than 3 months in other part of the body; hence the effect of pain condition on RNA transcription profiles could be excluded. Clinical parameters such as the degree of pain free opening and maximum unassisted opening as well as the duration of TMD symptoms including TMJ noise and difficulties in opening and/or closing the mouth were evaluated. Participants were diagnosed following the Diagnostic Criteria for Temporomandibular Disorder Axis ITo minimize the effect of circadian variation and menstruation cycle, peripheral blood was collected from all participants between 9:00 a.m. and 11:00 a.m. during their mid-luteal phases. For each individual, 3 ml of peripheral blood was put into a PAXgene Blood RNA tube vacutainer tube . PBMC from the venous blood from all participants were isolated using Ficolle-Paque PLUS (Sigma-Aldrich). TRIzol reagent  was used to isolate the total RNA from PBMC. Genomic DNA contamination was removed using RNase-free DNase I. The amount of total RNA was measured using NanoDrop 2000  and the integrity and quality of total RNA samples were analyzed using an Agilent Technologies 2100 Bioanalyzer . All samples passed quality control RNA integrity analysis (RIN ≥ 7). cDNA libraries were constructed using the TruSeq Stranded Total RNA Sample Preparation Kit  according to the manufacturer’s protocol. To produce 100 bp paired-end reads, the total RNA was sequenced using the Illumina NovaSeq 6000 system . After sequencing, the indexed samples were demultiplexed before the generation of FASTQ files for analysis and assessed by FastQC version 0.11.7.Using HISAT2 version 2.1.0 with the best score matches reported for each read, all libraries were aligned to hg19 assembly of the human genome. The mapped reads were assembled using STRING Tie version 1.3.4d. Inter-gene expression comparisons were based on calculated fragments per kilobase of transcript per million (FPKM) mapped reads.2 (fold change) were calculated. The DEGs with an adjusted P ≤ 0.05 and log 2 (fold changes) ≤ -1 or ≥ 1 were determined. Using heatmap function, the hierarchical clustering of the expression profiles of detected DEGs was conducted.The expression level was normalized by calculating FPKM mapped reads. For DEG analysis, the value of log http://string-db.org, version 11.0), gene ontology (GO) pathway enrichment analysis was conducted to compare gene transcription patterns between RNAOA and CON and assess the functional association between encoded proteins. False discovery rate (FDR) adjusted P values were calculated for each enriched biological pathway and the threshold was set to an adjusted P < 0.01. Gene set enrichment analysis  was used to compare gene dysregulation patterns in RNAOA with those in CON.Using online tool STRING analysis . Using IPA, biological processes, canonical pathways, and networks of analysis were analyzed. An enrichment score measures the overlap of observed and predicted gene sets. A z-score assesses the match of observed and predicted regulation patterns, serving as a predictor of the activation state of the identified molecule.The threshold was set to an unadjusted −(ΔCt), and the fold change was calculated by the formula 2−(ΔΔCt). The qRT-PCR values were normalized using the average of the expression of the reference gene, GAPDH. Finally, the averaged fold ratios from the reference housekeeping gene were used as the relative mRNA level. Each experiment was conducted in triplicate. Two reactions, one without template and one without reverse transcriptase were also performed.To validate the differential expression, gene expression levels were examined using quantitative real time polymerase chain reaction (qRT-PCR). qRT-PCR reactions were conducted via the Step ONE Plus  using SYBR premix EX Taq II , and cDNA was synthesized using 1 μg of mRNA. The gene expression results were obtained using the formula 2P < 0.01 for encoded protein functional network analysis (FNA). The threshold was set to an unadjusted P < 0.05 to fully explore the results by IPA. To compare gene expression levels obtained by qRT-PCR between RNA OA and CON, Mann–Whitney U tests were utilized. The level of significance was set at P < 0.05.The differences in demographic features and TMD characteristics between CON and RNAOA were evaluated using Mann–Whitney U tests. The differences in gene expression patterns were defined as fold changes. Statistical analysis was achieved by setting the change in gene expression threshold to an unadjusted P = 0.268) and body mass index (P = 0.740) did not show statistical significance between CON and RNAOA. Participants in RNAOA showed 16.4 ± 28.0 months of TMD symptom duration and the amount of pain free opening and maximum unassisted opening showed a significant difference between CON and RNAOA (P = 0.036) (Table The differences in age (P < 0.05). A total of 41 genes were expressed significantly differentially between RNAOA and CON with 17 genes upregulated and 24 downregulated in the RNAOA (log 2 (fold change) ≥ 1 or ≤ -1, P < 0.05)  were assessed using the STRING portal . A total of 16 GO terms (P < 0.01) including three molecular and 13 biological terms were annotated by the GO function of molecular function and biological process  were observed: (1) HLA-C, HLA-F, TNFRSF25, CCHCR1, FCER1A, MS4A2, and CPA3, (2) FGFR2, EREG, CXCL8, and HRH4, and UTS2 (3) FCGR3B and TNFRSF8, and (4) IL11RA and IL13RA1 | > 1. IPA identified 20 annotated categories of diseases and functions that were significantly upregulated (z-score > 2.0) and included 13 of the DEGs from the study. The 5 top canonical pathways were associated with the role of natural killer cells, communication between innate and adaptive immune cells, and T cell signaling (Table IPA was carried out on the transcriptome dataset with significance set at P = 0.030), CXCL8 (P = 0.022), and IL11RA transcripts (P = 0.018)  Fig. .Figure 53. The progression of the TMJ OA may accompany compromised masticatory function and altered craniofacial morphology, which could affect an individual’s quality of life24. However, to date, the exact pathophysiology of TMJ OA particularly in young patients and clear molecular mechanisms of the development of TMJ OA have not yet been elucidated, so far. Immune dysfunction has been considered as one of the main etiological factors which may result in bony destruction in patients with OA from other joints13, but studies which clearly elucidate the immunological background of TMJ OA especially in young patients are sparse. RNA-seq is an effective tool for clarifying molecular mechanisms in genetic levels and could provide the novel therapeutic targets involved in a certain condition19. To the best of the knowledge, no study ever attempted to reveal the immune related etiology of TMJ OA using RNA-seq technology. Consequently, the aim of the present study was to investigate the role of immune dysfunction by analyzing transcriptional profiles of PBMC and search for new therapeutic targets for management of TMJ OA in young patients.Early onset of diseases, especially during pubertal phases and age of early 20 s is a unique feature of the TMJ OA25 and relationships between this altered innate immune function, T cell and B cell responses, and cartilage and bone degradation have been reported27. Aforementioned results from IPA demonstrated associations among various autoimmune disorders, including autoimmune thyroid disease, systemic lupus erythematosus (SLE), and occurrence of TMJ OA. Previous studies already have mentioned the high prevalence of temporomandibular disorders in patients with autoimmune thyroid disorders or SLE30. Even though severe condylar resorption in patients with rheumatoid arthritis or juvenile idiopathic arthritis has been detected31, few reports ever mentioned the bony changes of condyles in other autoimmune diseases such as autoimmune thyroid disorders or SLE. Because patients with autoimmune disorders were excluded from the present study, clear associations between autoimmune thyroid disorders or SLE and TMJ OA could not be clarified. Even though, previous studies which dealt with the associations between innate immunity and OA progression focused on increased autoreactivity related to the aging process, the role of altered innate immune function in bony destruction of TMJ condyles even in young females could be assumed through this study.Declined acquired immune responses accompanied by increased autoreactivity have been detected in the elderly32. The results from the present study demonstrated the increased expression levels of chemokine (C-X-C motif) ligand 8 (CXCL8), the involvement of signal transducers and activators of transcription 3 (STAT3) pathway, and cytokine mediated immune response in the development of TMJ OA in young females. Interleukin-8/CXCL8 (IL-8/CXCL8) has been found to be an attractant for neutrophils and a population of lymphocytes33. The increased level of IL-8 in synovial fluid from the knee OA and TMD patients with disc displacement has been reported37, but the mechanisms of IL-8 in subchondral bone changes in TMJ have not been clearly revealed. One study has reported elevated serum levels of CXCL8 and expression levels of STAT3 in knee OA patients38. This study suggested that CXCL8 may inactivate the mitosis of chondrocytes and further aggravate OA progression and indirectly induce the Janus kinase (JAK)/STAT3 signaling in chondrocytes. Although the specific molecular mechanisms of CXCL8 and STAT3 could not be revealed in the present results, the associations between CXCL8, STAT3 signaling, and subchondral bony destruction of TMJ condyles in young females could be suspected.Accumulating evidence suggested the involvement of inflammatory and immune responses in the pathogenesis of OA41. One animal study suggested the molecular mechanisms of the role of CD8 + T cells in the OA process that CD8 + T cells were activated once OA had been initiated and cartilage degeneration occurred more slowly in CD8 + T cell knockout mice than in wild-type42. The results from this study also showed the relationships between the occurrence of TMJ OA and T cell activity through increased expression levels of genes related to Tec kinase signaling, nuclear factor of activated T cells (NFAT) regulation, and OX40 signaling. Furthermore, cytotoxic T lymphocyte mediated apoptosis and CTLA4 signaling pathway which have an association with cytotoxic T cell function, seem to have roles in the incidence of TMJ OA. A previous study has focused on the role of T helper cell in synovial fluid in the subchondral bony changes in TMJ OA patients16, but no study ever attempted to clarify altered cytotoxic T cell activities in blood in patients with TMJ OA.The peripheral blood of patients with OA have been analyzed and revealed that patients with OA have shown altered levels of CD8 + T cells and more cytotoxic profiles in comparison with healthy controlsDue to cross-sectional study design, the causal relationships between altered RNA transcriptional profiles and development of TMJ OA could not be derived from this study. The altered transcriptional profile would be result from TMJ bony destruction, and also abnormal immune-modulation function could be one of the main etiological factors of the TMJ OA. Because TMJs are small peripheral joints, the bony changes of the TMJs may not induce prominent changes in the systemic transcriptional profiles in the peripheral blood. However, regarding the statistically significant differences in the selected pathway and detected genes and sample number over 30, those differences could not be results just from chance.To the best of our knowledge, the present study is the first study which attempted to reveal the genetic and molecular background of TMJ OA in young patients using the RNA-seq technology. Several previous reports which dealt with the systemic immune dysfunction and the development of OA have focused on the role of the aging process and the senescence of immune cells. In addition, most of the studies which analyzed the relationships between levels of the immune-modulating agents in blood and OA dealt with weight bearing axial joints such as the knee and hip not with peripheral joints. Aforementioned results showed that altered systemic immune function could have a role in the development of arthritic conditions even in young patients with OA from small peripheral joints such as TMJ. However, the present study still has several limitations, first of all, RNA-seq was conducted using only PBMC, not using the synovial tissue. However, because TMJ is small joint, synovial fluid collecting process which accompanies with puncturing process inevitably causes iatrogenic inflammation in the small joint space and this may affect the RNA transcriptional profiles. Furthermore, the aim of this study was to figure out the immune-related pathophysiology of TMJ OA in young females, so adopting peripheral blood would be essential to reveal the purpose of this study. Secondly, only female participants were included. Even though previous studies confirmed the female preponderance of TMJ OA, the absence of male participants would limit the understanding of the genetic etiology of the TMJ OA. Thirdly, owing to the cross-sectional characteristics of the study, the information regarding the changes in the transcriptome profiles in accordance with the progression or recovery of TMJ OA could not be provided. Finally, the lack of control with inflammatory arthritis including juvenile idiopathic arthritis and rheumatoid arthritis could give limited information. However, no study which tried to reveal the immune-related pathophysiology of the TMJ OA based on transcriptional profiles of the peripheral blood have been investigated. Future prospective RNA-seq studies with large samples including both male and female participants and another control with inflammatory arthritis would be necessary.In conclusion, in the present study, young female TMJ OA patients showed alterations in the blood immune cell expression and some of the changes may reflect inflammation, autoreactivity, and altered T cell functions. For proper management and successful treatment of TMJ OA in young females, future research regarding immune-modulation based therapy would be warranted.


Objectives: Hypothenar hammer syndrome (HHS) is a rare vascular disease caused by blunt trauma of the hypothenar region. The optimal therapeutic strategy remains debatably since no large comparative studies are available yet. We want to evaluate the effectiveness of intra-arterial thrombolysis on angiographic and clinical outcome parameters in patients with HHS by performing a systematic review of the existing literature.Methods: A literature search of PUBMED/MEDLINE and SCIENCE DIRECT databases was performed up to May 2021.Results: In total, 16 manuscripts with 43 patients were included in the systematic review. Intra-arterial thrombolysis led to angiographic improvement in 29 patients (67.4%) and to clinical improvement in 34 patients (79.1%). Deterioration of arterial perfusion or clinical symptoms after thrombolysis were absent. Post-interventional complications were reported in only one patient (2.3%) without any bleeding complication. Logistic regression analyses demonstrated that a combined administration of fibrinolytics and heparin was associated with a significantly improved arterial patency  without significant amelioration of clinical symptoms . The use of rt-PA compared to other fibrinolytics and a prolonged thrombolysis duration of more than 24 h did not show statistically significant effects. Intra-arterial thrombolysis was significantly less effective in patients who had undergone thrombolysis with a delay of more than 30 days regarding clinical improvement .Conclusions: Intra-arterial thrombolysis with a combination of fibrinolytics and heparin is an effective and safe therapeutic option in patients with acute HHS. Hypothenar hammer syndrome (HHS) represents a rare ischemic disease of the hand and digital arteries which is typically caused by mechanical trauma to the distal ulnar artery at the hypothenar region –3. UlnarSymptoms of HHS vary from subtle digital discoloration and cold intolerance to severe digital and hand ischemia with threatening digital gangrene. Diagnostics in HHS seem to be well-established including physical status with Allen's test, digital acral plethysmography, color-coded ultrasound and angiography. Furthermore, laboratory testing may be considered to exclude potential immunological or hematological diseases, especially in atypical presentations of HHS . CompareThe aim of this study was to evaluate the effectiveness of intra-arterial thrombolysis on clinical and angiographic outcome parameters in patients with diagnosed HHS by performing a systematic review of the scientific literature on this topic.A systematic search of the literature was performed to evaluate in adults with HHS the effectiveness of intra-arterial thrombolysis on clinical and angiographic outcome parameters qualitatively and quantitatively. Primary endpoints were changes of clinical symptoms and thrombotic depositions after intra-arterial thrombolysis. Secondary endpoints were the occurrence of post-interventional complications, the effectiveness of a combined administration with fibrinolytics and heparin, as well as the influence of different fibrinolytic agents, duration of intra-arterial thrombolysis and of patients' delay to presentation on clinical and angiographic outcome. The systematic review was performed in accordance with the PRISMA 2009 guidelines while no review protocol was prepared . We pre-An electronic literature search of the databases PUBMED/MEDLINE and SCIENCE DIRECT was conducted in May 2021. The keywords “hypothenar hammer syndrome” together with “thrombolysis,” “fibrinolysis,” “fibrinolytics,” “urokinase,” “streptokinase,” “alteplase,” “tenecteplase,” “reteplase,” or “rt-PA” were used. We considered publications in English, French, and German, screened all abstracts and retrieved the full printed manuscript of all abstracts with a possible reported use of thrombolytic therapy in HHS. Additionally, full printed papers were retrieved when abstracts were missing and from studies with a possible reported use of thrombolytic therapy in HHS, which were not detected by the search in PUBMED/MEDLINE and SCIENCE DIRECT but cited in one of the retrieved papers. All abstracts and full texts were evaluated independently by two investigators (P.J. and F.H.). Data were extracted from all eligible publications regarding patient characteristics, including age, profession or initial symptoms, endovascular specifications, including thrombolysis duration or used fibrinolytics, as well as clinical and angiographic outcome parameters. Clinical and angiographic changes after intra-arterial thrombolysis were qualitatively assessed into “complete relief of symptoms” or “complete arterial patency,” “improvement with mild symptoms” or “improvement with residual thrombotic depositions,” “no change of symptoms” or “no change of arterial patency,” and “deterioration of symptoms” or “deterioration of arterial perfusion.” Additionally, quantitatively assessed clinical and angiographic changes were recorded if assessment methods to quantify clinical or angiographic changes were reported in the retrieved manuscripts. Furthermore, numbers of affected hand and digital arteries with completely restored patency without thrombotic depositions after intra-arterial thrombolysis were recorded. All eligible publications were evaluated by a proposed tool for methodological quality assessment of case reports and case series using an aggregated quality score .The study was approved by the institutional review board (EK 28-260 ex 15/16) and all research was carried out according to the relevant guidelines and regulations.p-values < 0.05. The statistical analyses were performed using R version 3.6.1.Categorical parameters are presented as absolute and relative frequencies whereas for continuous parameters mean and standard deviations (SD) or median as well as range (minimum to maximum) are given. Since this systematic review consists of case reports and case series, individual patient data are available for most parameters. Therefore, simple logistic regression analyses to assess the influence of several risk factors regarding clinical and angiographic outcome was performed as univariable analysis. Clinical and angiographic outcome parameters were trichotomized into “any improvement,” “no change” and “any deterioration.” Results are presented as odds ratios (OR) and 95% confidence intervals (CI). Statistical significance was assumed for In total, 317 primary publications could be identified by the literature search using the matching keywords. Abstracts were screened for a possible reported usage of thrombolytic therapy in patients with HHS and full-texts of 64 papers were retrieved. After removing duplicates listed in both PUBMED/MEDLINE and SCIENCE DIRECT and publications that did not report the usage of a thrombolytic therapy, 14 eligible publications could finally be identified , 16–27. Within the 16 papers included in this review, 43 patients  were described who underwent intra-arterial thrombolysis due to HHS. Mean (SD) age was 47.7 (13.3) years (range 18–69 years) for 35 patients in this systematic review, while one study with eight patients reported only a mean age of 52 years for a larger study collective . All patAngiographic improvement of the arterial patency by intra-arterial thrombolysis was achieved in 29 patients (67.4%) and amelioration of the reported symptoms in 34 patients (79.1%). Intra-arterial thrombolysis led to completely patent digital arteries in 19 patients (44.2%), to a completely patent ulnar artery in seven patients (16.3%) and to completely patent palmar arches in one patient (2.3%), but failed to achieve patency in seven patients (16.3%). Information about arterial patency is missing for 15 patients (34.9%). Deterioration of clinical symptoms or arterial perfusion after intra-arterial thrombolysis was not reported. Quantitative assessment of clinical and angiographic changes was impossible due to absent reports of quantitative assessment methods in the respective retrieved manuscripts. Therefore, only qualitative assessment of clinical and angiographic changes could be performed. Details about patients' angiographic and clinical outcome after intra-arterial thrombolysis are listed in One patient (2.3%) developed an abscess of the puncture site, while bleeding complications were not reported after intra-arterial thrombolysis. In two patients (4.7%) with initial acral necrosis, necrotic tissue was surgically removed while in three other patients (7.0%) with acral necrosis no surgical intervention was necessary. Surgical bail-out procedure by bypass grafting was performed in two patients (4.7%) after intra-arterial thrombolysis due to absent clinical improvement. In one patient (2.3%), bypass grafting was performed due to aneurysm resection.p = 0.005] compared to the administration of fibrinolytics alone. However, combined administration of fibrinolytic agents and heparin did not lead to a significant improvement of clinical symptoms .Combined administration of heparin was performed in 27 patients (62.8%) of which a constant infusion rate was prescribed with 500 IU/h in two patients (4.7%), with 833 IU/h in nine patients (20.9%) and with enoxaparin 0.4 ml/h in one patient (2.3%). Of the remaining 15 patients (34.9%), heparin dosage was administered aPTT-adjusted achieving a target range of 60 s 13 times while the heparin dosage was unmentioned two times. Combined administration of fibrinolytic agents and heparin was associated with a significantly improved arterial patency  compared to urokinase.Recombinant tissue plasminogen activator (rt-PA) was administered in 21 patients (48.8%). Thereof, one study reported only the administration of 14.7 ml rt-PA in seven patients ; the remic agent . The usep = 0.222] nor of clinical symptoms  compared to a duration of 24 h or less. Intra-arterial thrombolysis was less effective in patients who had undergone it with a delay of more than 30 days regarding clinical improvement  compared to those patients who had undergone thrombolysis within 30 days. However, prolonged delay did not significantly influence angiographic improvement .The median duration of intra-arterial thrombolysis was 17 h (range 2–72 h) while one study reported only a thrombolysis duration of >11 h . AnotherHHS is a rare vascular ischemic condition caused by blunt trauma of the hypothenar region. Due to its rarity, treatment options are empiric and decisions regarding conservative treatment, intra-arterial thrombolysis or surgery depend on ischemia severity, symptom duration and vessel morphology. Although reviews reported benefits of intra-arterial thrombolysis in digital and hand ischemia, including patients with HHS, data on intra-arterial thrombolysis have been derived mostly from case reports using different thrombolytic methods and different reported outcomes , 30, 31.Discrepancy between improvement of clinical symptoms and patent arterial perfusion could be observed in this systematic review. Amelioration of clinical symptoms was established in 79.1% while an improved arterial perfusion with residual thrombotic depositions was achieved in only 67.4% after intra-arterial thrombolysis. Complete arterial patency was observed in only 11.6% whereas complete relief of symptoms was achieved in 32.6% of reported patients. This observation suggests that a decrease of thrombotic depositions, even without complete restoration of the arterial perfusion in digital and hand arteries, may be sufficient to provide adequate blood flow leading consequently to an amelioration of clinical symptoms. One possible explanation for this observation may be attributed to good collateral vessels of the hand, since distinct collateral vessels may also buffer ischemic symptoms in lower limb ischemia. Chronic collateral artery formation of the hand may contribute also to a benign prognosis in surgically and non-interventionally treated patients with HHS. Long-term results of surgical treatment in HHS reported relatively low patency rates although patients complained only mild symptoms and persisting symptoms occurred less frequently and less severely after 5-years follow-up period in patients with HHS treated conservatively –37. HoweThis systematic review demonstrated that a combined administration of fibrinolytic agents and heparin revealed significant higher angiographic patency rates but without significant improvement of symptoms. Nevertheless, a combination therapy with fibrinolytics and heparin should be preferred for treating patients with HHS due to a favorable risk-benefit ratio toward a potential improvement of clinical symptoms while the bleeding risk seem to be very low without any reported bleeding complication. Furthermore, this systematic review could demonstrate for the first time that intra-arterial thrombolysis in patients with HHS was less effective if administered with a delay of more than 30 days and that a prolonged thrombolysis duration of more than 24 h has no benefits on arterial patency or clinical outcome indicating that intra-arterial thrombolysis should be reserved for acute and subacute forms of HHS.Limitations of this study are firstly the small number of patients, namely only 43, and secondly that only case reports and small case series could be included. However, this systematic review describes the largest cumulative patient cohort in HHS treated with intra-arterial thrombolysis yet. Another limitation is that long-term outcome data were only rarely reported and no analysis could be performed. Further limitations may be absent comparisons of intra-arterial thrombolysis to surgical and conservative treatment options as well as that clinical and angiographic outcome parameters could not be assessed by quantitative measurement methods.In conclusion, the use of endovascular thrombolysis in patients with acute and subacute HHS with persisting symptoms of no longer than 30 days is associated with high clinical benefit, high arterial patency rate and low post-interventional complications. Overall, however, since randomized trials or at least large case-control study comparing endovascular, surgical and conservative medical treatment options in HHS are absent, current treatment in HHS is still rather empiric.The original contributions presented in the study are included in the article/PJ and FH contributed to data collection, conceptualization, drafting, and reviewing of the manuscript. FH contributed to study design. GP and AB contributed to statistical analysis and reviewing of the manuscript. PR, VM, and KG contributed to drafting and reviewing of the manuscript. MB contributed to supervision of the manuscript. All authors approved the submitted version.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.


We hypothesized that these lesions would be associated with severe cerebral amyloid angiopathy. Ten cases were selected from the long-term follow-up study of patients who enrolled in the first clinical trial of active amyloid β immunization with AN1792 for Alzheimer’s disease. Eleven matched non-immunized Alzheimer’s disease cases from an independent brain brank were used as ‘controls’. Formalin-fixed occipital brain slices were imaged at 7 T MRI to screen for haemorrhagic lesions . Samples with and without haemorrhagic lesions were cut and stained. Artificial intelligence-assisted quantification of amyloid β plaque area, cortical and leptomeningeal cerebral amyloid angiopathy area, the density of iron and calcium positive cells and reactive astrocytes and activated microglia was performed. On ex vivo MRI, cortical superficial siderosis was observed in 5/10 immunized Alzheimer’s disease cases compared with 1/11 control Alzheimer’s disease cases (κ = 0.5). On histopathology, these areas revealed iron and calcium positive deposits in the cortex. Within the immunized Alzheimer’s disease group, areas with siderosis on MRI revealed greater leptomeningeal cerebral amyloid angiopathy and concentric splitting of the vessel walls compared with areas without siderosis. Moreover, greater density of iron-positive cells in the cortex was associated with lower amyloid β plaque area and a trend towards increased post-vaccination antibody titres. This work highlights the use of ex vivo MRI to investigate the neuropathological correlates of haemorrhagic lesions observed in the context of amyloid β immunotherapy. These findings suggest a possible role for cerebral amyloid angiopathy in the formation of haemorrhagic amyloid-related imaging abnormalities, awaiting confirmation in future studies that include brain tissue of patients who received passive immunotherapy against amyloid β with available in vivo MRI during life.Haemorrhagic amyloid-related imaging abnormalities on MRI are frequently observed adverse events in the context of amyloid β immunotherapy trials in patients with Alzheimer’s disease. The underlying histopathology and pathophysiological mechanisms of haemorrhagic amyloid-related imaging abnormalities remain largely unknown, although coexisting cerebral amyloid angiopathy may play a key role. Here, we used  et al. report haemorrhagic lesions on ex vivo MRI in brain tissue samples of Alzheimer’s disease patients who received anti-amyloid immunotherapy during life. These lesions were associated with severe leptomeningeal cerebral amyloid angiopathy, suggesting a possible role for cerebral amyloid angiopathy in the formation of haemorrhagic amyloid-related imaging abnormalities.Scherlek   In this framework, intervention strategies have focused on anti-Aβ immunotherapy to halt a detrimental cascade of events. Long-term follow-up studies of the first active immunization trial  demonstrated that Aβ can be effectively removed from the brain.3 Subsequent passive immunotherapy trials similarly demonstrated Aβ clearance, culminating most recently in the US Food and Drug Administration’s approval of aducanumab for the treatment of Alzheimer’s disease. One of the major obstacles in the development of successful passive anti-Aβ immunotherapies has been the occurrence of adverse events including amyloid-related imaging abnormalities (ARIA) on brain MRI. ARIA can be observed in the form of signal hyperintensities on fluid-attenuated inversion recovery (FLAIR) thought to represent acute/subacute vasogenic oedema and/or sulcal effusion termed ARIA-E, and signal hypointensities on T2*-weighted gradient echo (GRE) MRI thought to represent haemosiderin deposits  termed ARIA-H.4–6 When ARIA presents with symptoms, they can be managed with steroids and discontinuation of the anti-Aβ immunotherapy.7 The first active immunization trial against Aβ was halted because of the development of meningoencephalitis in 6% of participants, which shared similarities with ARIA although the term had not been introduced yet at the time.9 Subsequent passive immunotherapy trials have reported ARIA in up to 47% of participants receiving the highest antibody doses.11 Most recent data from the Phase 3 aducanumab trials indicated that ARIA-E was observed in 35% and ARIA-H in 19% (microbleeds) and 15%  of patients treated with the highest dose (10 mg/kg).12A prominent feature of Alzheimer’s disease is the accumulation of the amyloid β (Aβ) peptide in the brain in the form of plaques and cerebral amyloid angiopathy (CAA).13 Approximately 50% of patients with Alzheimer’s disease have moderate-to-severe CAA on post-mortem examination of the brain.14 The pathophysiology of CAA is postulated to involve a feed-forward cycle of impaired perivascular Aβ drainage, subsequent accumulation of Aβ in the walls of cortical arterioles and leptomeningeal arteries, smooth muscle cell degeneration, impaired vascular compliance and further Aβ accumulation. Eventually, this results in vessel wall breakdown, extravasation of plasma proteins into the brain and haemorrhages.1 Brain haemorrhages in the form of lobar microbleeds, cortical superficial siderosis and symptomatic lobar intracerebral haemorrhage are the hallmark neuroimaging manifestations of CAA and form the basis of the diagnostic criteria for CAA during life.16 A subset of patients with CAA presents with spontaneous ARIA-like imaging abnormalities and clinical symptoms in the form of vasogenic oedema and haemorrhages, termed CAA-related inflammation. Analysis from Pittsburgh compound B (PiB)-PET imaging studies and CSF samples suggests that CAA-related inflammation may be the result of a spontaneous anti-Aβ immune response and immune-mediated Aβ removal from the brain.18 It remains unknown whether similar mechanisms play a role in the formation of ARIA in Aβ immunotherapy trials in Alzheimer’s disease patients.20 So far, neuropathological investigations of ARIA have been limited due to the scarcity of appropriate autopsy cases. To address this knowledge gap, we performed ex vivo MRI in formalin-fixed brain tissue samples of cases that underwent active immunization with AN1792 against Aβ to screen for possible ARIA-H and to perform a detailed neuropathological investigation of the underlying tissue and vascular abnormalities. Our investigation was limited to haemorrhagic lesions as the transient nature of ARIA-E precludes the assessment of these lesions ex vivo.The neuropathological associations and pathophysiological mechanisms of ARIA are currently unknown, although the presence of CAA has been suggested as a major contributing factor.3 Ethical approval was obtained through the Southampton and South West Hampshire Local Research Ethics Committees (REC reference 075/03/w). Brain tissue from 11 matched  non-immunized Alzheimer’s disease cases was included as ‘controls’ (cAD cases) from the South West Dementia Brain Bank (REC reference 18/SW/0029). One iAD case (Case #1) had documented ARIA during life8 and as such was considered a positive control case. Two out of 12 iAD cases received placebo, of which one (Case #14) proved not to have Alzheimer’s disease on neuropathological examination. As such, the placebo cases were excluded, and we considered the cAD cases to be the negative controls in this study.Twelve cases with well-preserved formalin-fixed brain tissue from the long-term follow-up study of patients who enrolled in the first clinical trial of active Aβ immunization for Alzheimer’s disease  and provided consent for brain donation were included (iAD cases).Six additional non-Alzheimer’s disease control cases were included to control for long-term formalin fixation .ex vivo MRI scanning. The occipital lobe was selected as CAA tends to be more severe in the posterior parts of the brain.From each case, one intact formalin-fixed coronal brain slice from the occipital lobe from a single cerebral hemisphere was selected for in vivo MRI was added to the study.21 From this case, an intact formalin-fixed coronal brain slice from the occipital and frontal lobe from a single cerebral hemisphere was selected for ex vivo MRI scanning.For comparison, formalin-fixed brain tissue of a case from the Massachusetts General Hospital stroke research centre with a clinical diagnosis of probable CAA and evidence of disseminated cortical superficial siderosis on Detailed case characteristics are presented in 2*-weighted GRE sequence  and a T2-weighted turbo spin echo (TSE) sequence .Three-to-four slices were simultaneously scanned for each MRI scan session. Slices were submerged in 10% formalin at room temperature in a glass container that fit in the head coil of the MRI scanner. Care was taken to avoid air bubbles by gently shaking the tissue. Scans were acquired overnight on a whole-body 7 T MRI scanner  with a custom-built 32-channel head coil. The scan protocol included a Tex vivo MRI before, two raters  screened the acquired MR images first for the presence of haemorrhagic lesions. Hypointense signal abnormalities suggestive of cortical superficial siderosis22 were noted on the GRE scans in the form of curvilinear cortical hypointensities in both the positive control iAD case (Case #1) and several other iAD cases (but not the cAD cases). In addition, we noted occasional rarefaction of the juxtacortical white matter in the form of confluent hypointense abnormalities in iAD cases. No notable differences between iAD and cAD cases were observed in any of the other neuroimaging features on the TSE scans, such as ischaemic lesions, white matter hyperintensities or enlarged perivascular spaces. After this initial screening, the GRE MRI scans were de-identified and re-assessed by two raters  blinded to treatment status and clinical information. The inter-rater agreement for the assessment of focal or disseminated cortical superficial siderosis was moderate (κ = 0.515), and for widespread (affecting >50% of the tissue) white matter rarefaction was perfect (κ = 1.000). Discrepancies were resolved in a consensus meeting, after which a final rating was established.Since ARIA-H has not been described on ex vivo MRI and cut to fit a standard tissue cassette (measuring ∼30 × 25 × 4 mm). In the cAD and non-Alzheimer’s disease cases, at least one area was sampled at the level of the calcarine cortex, and additional areas if abnormalities were observed on MRI. Collectively, from the iAD cases, a total of 10 samples were taken from areas with haemorrhagic lesions suggestive of ARIA-H (in the five cases with siderosis on MRI) and a total of 12 samples were taken from random areas without haemorrhagic lesions (in five cases without and two cases with siderosis on MRI). From the cAD cases, a total of 16 samples were taken. From the non-Alzheimer’s disease cases, a total of seven samples were taken. Samples were processed and embedded in paraffin and cut in 6 μm thick serial sections on a microtome.In the iAD cases, areas with and without haemorrhagic lesions were sampled guided by 2O2 (20 min), treatment with formic acid , antigen retrieval in heated citrate buffer  and AT8, 20 min), blocking with normal horse or goat serum (1 h), overnight incubation with the primary antibody at 4°C, incubation with a biotinylated mouse or rabbit secondary antibody , followed by incubation with a mixture of avidin (A) and biotinylated HRP (B)  and development with 3,3′-diaminobenzidine (Vector Laboratories). Sections were counter stained with haematoxylin (10 s), dehydrated through graded ethanol series and cover slipped with Fisher Chemical Permount mounting medium. In between steps, sections were washed with tris-buffered saline.Adjacent sections were stained with haematoxylin and eosin (H&E), Luxol fast blue H&E (for myelin), Perls’ Prussian blue (for iron) and Von Kossa  using standard histology protocols. Immunohistochemistry was performed against pan-Aβ , hyperphosphorylated tau , glial fibrillary acidic protein , phagocytic microglia  and fibrin(ogen) . The standardized immunohistochemistry protocol included deparaffinization and rehydration of the tissue through xylene and graded ethanol series, blocking of endogenous peroxidase with 3% HAll stained sections were scanned on a NanoZoomer whole slide scanner  using a 20× objective, and the obtained digital images were assessed using the NDP.view2 viewing software (version 2.7.25).23 (ii) number of microbleeds on H&E and Perls’,23 (iii) degree of concentric splitting of the wall of leptomeningeal blood vessels on H&E and Aβ-stained sections as absent, mild, moderate, severe (0–3)24 and (iv) tau severity on AT8-stained sections as absent, mild, moderate, severe (0–3). Final ratings were established in a consensus meeting.Sections were de-identified and assessed by two independent raters (A.A.S. and S.J.v.V.) blinded to MRI findings, treatment status and clinical information, for the following: (i) number of microinfarcts on H&E and GFAP,25 Briefly, digital images were uploaded to the platform, after which several convolutional neural networks were trained on manual annotations (drawn by AAS) of histopathological features of interest on a representative subset of sections. Once fully trained, the AI-models were applied to all sections (one section per stain per area). We obtained the following quantitative measurements: (i) percentage cortical tissue area occupied by Aβ plaques, (ii) percentage cortical tissue area occupied by CAA, (iii) percentage leptomeningeal area occupied by CAA, (iv) density of iron-positive deposits within the cortex, (v) density of calcium positive cells within the cortex, (vi) cell density of reactive (GFAP-positive) astrocytes within the cortex, (vii) cell density of activated/phagocytic (CD68-positive) microglia in the cortex and (viii) percentage cortical tissue area occupied by fibrin-positive vessels.In addition, we obtained artificial intelligence (AI)-assisted quantitative measurements using the online platform Aiforia®. The methodology is described in detail elsewhere.ex vivo MRI, the presence or absence of ex vivo MRI abnormalities was compared between iAD and cAD cases with χ2 tests for proportions. Clinical and general neuropathological observations were compared between iAD cases with and without the presence of haemorrhagic lesions on ex vivo MRI with Mann–Whitney U-tests for non-parametric data. Histopathological and vascular alterations between samples from cAD and iAD cases and samples with and without haemorrhagic lesions from iAD cases were compared with Mann–Whitney U-tests and χ2 tests. Correlation analyses were performed with Spearman correlations. Cohen’s kappa was used to assess inter-rater reliability. All statistical analyses were performed with IBM SPSS version 22 and graphs were created with GraphPad Prism version 9.0.2. P-values of <0.05 were considered statistically significant. Statistics were performed on the number of samples rather than the number of cases.To assess whether haemorrhagic lesions resulting from immunotherapy could be detected on The original data that support the findings of this study are available from the corresponding author upon reasonable request.A total of 10 iAD cases  and 11 cAD cases  were included . For theex vivo GRE 7T MRI scan of iAD Case #1, who had documented ARIA during life 12 months before death,8 curvilinear hypointense abnormalities resembling cortical superficial siderosis were observed .On the observed . SimilarOne microbleed was found in one of the cAD cases  .On the = 0.476) . Corticaex vivo MRI (iAD Case #17). Similar histopathological abnormalities were not present in the cAD cases, except for occasional extracellular calcium deposits in the cortex, which may be the result of long formalin fixation   .25 we found a greater density of intracellular iron and calcium deposits in the cortex in iAD cases (n = 22 samples) compared with cAD cases (n = 16 samples) . Aβ plaque area  and tau severity  were both significantly lower in iAD compared with cAD samples, which is in line with previous reports in this cohort3  and leptomeningeal CAA area  did not differ between iAD (n = 22 samples) and cAD cases (n = 16 samples). Moreover, severity of concentric vessel wall splitting (which was assessed semi-quantitatively) was comparable as well   .ex vivo MRI in the iAD group. We found a greater density of iron deposits in areas with (n = 10 samples) compared with areas without haemorrhagic lesions (n = 12 samples) , but not intracellular calcium deposits . Aβ plaque area  and tau severity  did not differ between areas with and without haemorrhagic lesions  on  lesions .n = 10 samples) and without haemorrhagic lesions (n = 12 samples) . However, we found greater leptomeningeal CAA area  as well as degree of concentric vessel wall splitting  in areas with compared with areas without haemorrhagic lesions .Regarding vascular alterations, percentage cortical CAA area was comparable between areas with . Although there was no overall difference between the presence of microinfarcts in areas with and without haemorrhagic lesions , 17/21 microinfarcts were observed in iAD Case #1 in areas with haemorrhagic lesions on ex vivo MRI . Moreover, higher post-vaccination anti-AN1792 antibody titres were associated with lower Aβ plaque area  and tended to be associated with greater density of iron deposits in the cortex   . Collectρ = 0.672, P = 0.001) and phagocytic microglia in the cortex   .ex vivo MRI in cases who received active immunization against Aβ during life. The ex vivo MRI and histopathological characteristics were consistent with cortical superficial siderosis, a known manifestation of severe CAA.21 Neuropathological examination confirmed locally severe vascular pathology of the leptomeningeal blood vessels, including greater vascular Aβ deposition and vessel wall splitting. The severity of cortical superficial siderosis was associated with lower Aβ plaque area and higher post-vaccination antibody titre levels. Finally, we observed greater presence of reactive glial cells in iAD cases with longer survival times.This study resulted in several main findings. We observed haemorrhagic lesions on in vivo MRI, we were not able to verify whether the haemorrhagic lesions on ex vivo MRI indeed corresponded to ARIA-H detected during life. Of note, similar hypointense signals on ex vivo MRI have been observed in post-mortem cases with early-onset Alzheimer’s disease26 and were observed in our current study in one non-Alzheimer’s disease control case. Moreover, another ex vivo MRI study in CAA patients identified similar hypointense stripes in the cortex, in these cases reflecting severe ferrugination in the form of iron and calcium depositions in cortical blood vessels.27 Taken together, the ex vivo MRI findings in iAD cases are not specific, although the underlying neuropathology in these areas  was suggestive of ARIA-H.Without 6 Two subtypes have been defined based on in vivo MR imaging characteristics; ARIA-E  and ARIA-H .6 Both subtypes were initially observed in active immunization trials and were defined as subacute meningoencephalitis,9 and have now also been reported in multiple passive immunotherapy trials with rates of up to ∼40% in the Phase 1b and Phase 3 aducanumab12 and Phase 2 donanemab trials.28 ARIA is usually asymptomatic with symptoms reported in 1–26% of patients with ARIA-E.12 Notably, the increased rate of ARIA-H in patients treated with passive immunotherapy as compared with placebo is more modest than differences in ARIA-E29, an effect largely driven by higher rates of pre-existing microbleeds in Alzheimer’s disease cases. In our study, we observed abnormalities suggestive of ARIA-H on ex vivo MRI in 5/10 iAD cases as compared with 1/11 cAD cases. Important risk factors for the development of symptomatic ARIA include immunotherapy dose, APOE ε4 allele status, and baseline number of cerebral microbleeds, with higher doses and/or faster up-titration of antibody and two copies of the APOE ε4 allele associated with increased incidence of ARIA.1 We did not find a relationship between haemorrhagic lesions and APOE ε4 status in the current study, which may be because all iAD cases had at least one allele as well as the modest sample size.The term ARIA refers to a spectrum of image abnormalities that can be observed throughout the natural course of Alzheimer’s disease and CAA and in the setting of amyloid-modifying therapeutic approaches.APOE ε4 allele status and number of microbleeds as well as similarities between ARIA- and CAA-related inflammation. Additionally, prior neuropathology findings have suggested increased CAA in iAD cases as well as increased concentric vessel wall splitting (i.e. lumen within a lumen appearance) detected in the frontal, temporal and parietal lobes.31 We did not observe similar differences in our study between iAD and cAD cases. As we included brain slices from the occipital lobe, in which more severe CAA is typically observed, we suspect that any relative increase in CAA in the iAD cases may not have been detectable because of the higher baseline CAA severity in cAD cases in these occipital areas.CAA has been proposed as a potential driver of ARIA formation, supported by shared risk factors between CAA and ARIA including in vivo T2-weighted imaging.8In vivo T2*-weighted imaging was not available for this patient, which precluded us from verifying the presence of cortical superficial siderosis or other types of ARIA-H post-vaccination. An extensive neuropathological study of this case was previously published, demonstrating regions with likely Aβ plaque removal with relative persistence of CAA as well as T-lymphocyte meningoencephalitis.8 Of note, this was the only case in this cohort observed to have fulminant meningoencephalitis at autopsy.3 This case had the shortest interval between first immunization and death, possibly reflecting the severity of the adverse event. However, whereas other cases who received active immunization did not have any evidence of active meningoencephalitis at autopsy, neuropathological examination did provide evidence for Aβ plaque removal and increased CAA.3Our ‘positive control’ (iAD Case #1) had symptomatic ARIA-E during life after receiving active Aβ immunization, confirmed by in vivo MRI scans to verify whether these lesions indeed met the criteria for ARIA-H during life, our findings may not be generalizable. The MRI characteristics were suggestive of cortical superficial siderosis as frequently observed in cases with severe CAA and one of the subtypes of ARIA in Aβ immunotherapy trials. Cortical superficial siderosis in CAA patients represents the chronic phase of convexity subarachnoid haemorrhage and increases the risk of future haemorrhagic stroke.32 Our histopathological findings are in line with previous observations in CAA autopsy cases with documented cortical superficial siderosis during life and are indicative of bleeding from the leptomeningeal blood vessels in the context of severe CAA.21 Notably, we previously reported increased leptomeningeal  CAA severity and concentric splitting of the walls (i.e. lumen within a lumen appearance) of leptomeningeal vessels in areas with siderosis on histopathology,21 resembling our observations in iAD cases. Moreover, siderosis was associated with increased numbers of cortical microinfarcts, thought to be the result of secondary injury after leptomeningeal bleeding, which is in line with our findings in iAD Case #1.Our findings extend this literature by adding the first direct histopathological investigation of haemorrhagic lesions in the context of Aβ immunotherapy. Without available 30 resulting in vessel wall remodelling and fragmentation 31 and subsequent leakage of blood products into the underlying cortex. An alternative interpretation of our findings is that the intraneuronal iron depositions observed in iAD cases are the result of released iron from local Aβ plaques after successful Aβ plaque removal. Previous studies have indeed demonstrated that Aβ plaques contain iron.33–36 In this scenario, our observations would not be related to bleeding and would likely be irrelevant to ARIA-H as observed during treatment trials. Although we cannot rule out this explanation, it does not account for the observed association with severe leptomeningeal CAA and concentric vessel wall splitting. Future studies, including longitudinal in vivo imaging studies in mouse models are needed to tease out the individual steps and exact sequence of events in ARIA formation.37Although the cross-sectional nature of this investigation does not allow us to infer causes and consequences, potential mechanisms that could explain our findings in iAD cases are proposed as follows: perivascular drainage of Aβ from the cortex after immunization may have resulted in increased deposition of Aβ in leptomeningeal arteries,39 This observation is detected with the addition of cases with longer post-immunization survival times and thus could reflect that the response to ongoing neurodegenerative processes, such as tau pathology, is still progressing.13 Future studies are needed to determine the nature of these neuroinflammatory responses.We found that longer survival times were associated with increased number of reactive glial cells, while previous studies reported an overall downregulation of neuroinflammatory protein expression following Aβ removal.ex vivo GRE MRI in the iAD cases may reflect artefacts due to prolonged formalin fixation. A previous study reported similar-appearing granular neuropil changes on H&E in tissue that was fixed for more than 6 years.40 These alterations were also present in non-Alzheimer’s disease control cases, further suggesting these are irrelevant to ARIA-H. Moreover, similar abnormalities were not found in tissue from the same iAD cases embedded in paraffin after weeks of fixation. Importantly, Von Kossa staining of these samples embedded within weeks of fixation did demonstrate intracellular calcium deposits in the cortex in one of the most severely affected iAD cases (Case #7). The adjacent iron-stained section was also positive, but the signal was notably less strong than the iron-stained sections in the tissue of the same case after years of fixation. One explanation for this apparent mismatch may be that formalin fixation augments endogenous intracellular iron and calcium depositions. However, reports in the literature that have examined the effect of formalin fixation on the concentration of minerals, including iron, either suggest no effect,41 or significantly lower iron levels in formalin fixed compared with fresh tissue.42 Alternatively, the absence of in vivo or ex vivo MRI to guide sampling in previous studies may explain why these tissue alterations had not been observed before in this dataset and further underlines the importance of ex vivo MRI to bridge the gap between observations made during life and neuropathology. It is unclear why similar amounts of extracellular calcifications were not found in the cAD cases that were matched for years of fixation and stored under similar conditions as the iAD cases. We therefore cannot completely rule out the possibility that some of the white matter rarefaction in iAD cases reflects old oedema in the setting of ARIA-E.The observation of extracellular calcifications in the tissue with accompanying hypointensities on ex vivo MRI, an initial unblinded screen was performed prior to blinded assessment by the same rater. Given the small sample size, this may have resulted in unintentional bias. Furthermore, rating ex vivo MRI scans remains challenging due to distortions (e.g. air trapped between stacked slices), which was reflected by a moderate inter-rater agreement. Of note, all neuropathological examinations were performed in areas where both raters agreed on the presence or absence of siderosis on ex vivo MRI locally. Fourth, since we did not have access to autopsy samples of patients who underwent passive immunotherapy during life, our findings may not be generalizable to anti-Aβ antibody treatments and ongoing studies that rely on passive immunotherapy.Besides the abovementioned caveats, this study has several additional limitations. First, because of the small number of cases that were available, we considered individual tissue blocks as independent samples. This can be considered a limitation given the unequal distribution of samples across cases. Second, only occipital brain slices were included in this study and it remains unclear whether similar alterations occur in other parts of the brain in which CAA is generally less severe. Third, because of the lack of established rating criteria for ARIA-H on ex vivo MRI to screen for ARIA-H. With the abovementioned significant limitations in mind, our findings suggest that Aβ immunotherapy may result in chronic cortical superficial siderosis in the context of severe leptomeningeal CAA and vascular remodelling. Whether secondary tissue injury caused by cortical superficial siderosis such as cortical microinfarcts and neuronal mineralization may contribute to further cognitive decline in Alzheimer’s disease patients with ARIA-H needs to be assessed in future studies. Collectively, the screening, clinical management and ideally prevention of ARIA-H during Aβ immunotherapy trials are potentially relevant to limiting clinical deterioration, especially in Alzheimer’s disease patients with co-occurring CAA or APOE ε4 carriers who are at increased risk.43This study demonstrates the added value of using fcac021_Supplementary_DataClick here for additional data file.


Radiation-induced lung injury (RILI) is a potential complication of thoracic radiotherapy that can result in pneumonitis or pulmonary fibrosis and is associated with significant morbidity and mortality. The pathobiology of RILI is complex and includes the generation of free radicals and DNA damage that precipitate oxidative stress, endothelial cell (EC), and epithelial cell injury and inflammation. While the cellular events involved continue to be elucidated and characterized, targeted and effective therapies for RILI remain elusive. Sphingolipids are known to mediate EC function including many of the cell signaling events associated with the elaboration of RILI. Sphingosine-1-phosphate (S1P) and S1P analogs enhance EC barrier function in vitro and have demonstrated significant protective effects in vivo in a variety of acute lung injury models including RILI. Similarly, statin drugs that have pleiotropic effects that include upregulation of EC S1P receptor 1 (S1PR1) have been found to be strongly protective in a small animal RILI model. Thus, targeting of EC sphingosine signaling, either directly or indirectly, to augment EC function and thereby attenuate EC permeability and inflammatory responses, represents a novel and promising therapeutic strategy for the prevention or treatment of RILI. Radiation-induced lung injury (RILI) represents a dose-limiting toxicity associated with thoracic radiotherapy for which effective treatments remain non-existent. RILI encompasses a clinical spectrum of disease manifested early (6–12 weeks after radiation) by pneumonitis and later (at 6–12 months) by fibrosis associated with irreversible impairment of lung function . NonetheLung inflammatory syndromes, including RILI, represent a collection of clinical conditions of which a key feature is endothelial dysfunction and increased vascular permeability. The clinical consequences of these changes are impaired oxygenation and lung function associated with non-uniform radiographic changes. These characteristic changes in RILI support endothelial cell (EC) barrier dysfunction as a key determinative event, consistent with other forms of acute lung injury (ALI). Accordingly, strategies aimed at augmenting or restoring endothelial barrier function offer therapeutic promise and potential. Our lab and others have previously identified specific molecular targets including sphingolipids, as particularly promising in this regard –9.Sphingolipids, constituents of cell plasma membranes, mediate a variety of cell signaling events. Sphingosine-1-phosphate (S1P) and ceramide are two sphingolipids that have been implicated as mediators of inflammatory lung injury in response to radiation. While S1P is a pro-survival molecule, ceramide promotes apoptosis and its production is increased in response to cellular stress including that caused by radiation . S1P is Five G protein-coupled S1P receptor subtypes have been identified and are widely expressed on various cell types. S1PR1, S1PR2, and S1PR3 are predominantly expressed on EC but are coupled to different G proteins resulting in distinct downstream signaling effects . S1PR1 lSubsequently, S1P was found to be protective in pre-clinical ALI models as well , 17. In Our group has thoroughly characterized a murine RILI model that we have employed to study potential therapeutic strategies –21. BrieThus, the targeting of sphingolipid signaling as a therapeutic strategy to prevent or mitigate RILI can be broadly grouped into either direct or indirect approaches. The use of S1P or S1P analogs is indicative of approaches that directly augment sphingolipid pathway signaling. Alternatively, a variety of different strategies can be considered to indirectly augment sphingolipid signaling, for example, via upregulation of S1PR1 or modulation of SphK expression levels. These ideas are discussed in more detail below.Several reports have confirmed robust protective effects of S1P in animal models of ALI. However, the translational values of these observations are limited but serious safety concerns related to the use of S1P in patients. Specifically, S1P administration is associated with potential cardiovascular effects including significant bradycardia as well as reductions in cardiac output , 25. Accvβ3 antagonist and a near-infrared fluorochrome. Animals were injected with the probe 6 weeks post radiation and imaged 24 h later  with lung injury assessed at 6 weeks via BALF analyses. Notably, tyspinate had the most dramatic effect with both low and high dosing (0.01 or 0.1 mg/kg) resulting in a significant attenuation of RILI. In comparison, SEW was found to be protective with higher dosing (0.1 mg/kg) while no effect was observed with FTY720. Furthermore, evidence of protection was observed with all three agonists as assessed by lung histology as well as by VisEn FMT lung imaging . This technique allows for real-time imaging of microvascular leak in vivo utilizing a fluorescence imaging agent  that targets the vasculature as a selective nonpeptide small molecule integrin αWhile these experiments were largely proof of principle, they support the idea that S1P analogs could potentially be used to prevent or treat RILI. Importantly, treatment with these S1P analogs was well tolerated by the animals that received them with no adverse effects appreciated. Moreover, with respect to the potential clinical applications, unlike other ALI patients whose disease is largely unpredictable, the idea of pre-treatment strategies could easily be adapted and applied to patients undergoing radiation therapy, particularly those who may be deemed to be at increased risk for RILI.The statin drugs are a class of HMG CoA-reductase inhibitors that have been widely used for their ability to lower serum cholesterol levels and for their beneficial effects in patients with or at high risk for cardiovascular disease. These drugs, however, are also recognized to have pleiotropic effects independent on their lipid-lowering properties. Included amongst these are direct effects on EC signaling and vascular function. Arguably, with respect to effects just on EC alone these drugs could also said to have pleiotropic properties via inhibition of protein prenylation including effects on RhoGTPase activation, NADPH oxidase activation, and nitric oxide expression, as well differential expression of a variety of genes including mediators of actin cytoskeletal dynamics and inflammation, determinants of EC barrier function and vascular permeability, as well as effects on sphingolipid pathway components –31. IndeS1PR1 promoter activity that is associated with KLF2 recruitment to the S1PR1 promoter. Furthermore, this activation of the S1PR1 promoter is attenuated by KLF2 silencing (siRNA). The functional importance of these effects is evidenced by augmentation of S1PR1-mediated EC barrier augmentation by simvastatin that is reduced by KLF2 silencing as measured by changes in transendothelial electrical resistance [With respect to sphingolipids in particular, we previously reported that simvastatin upregulates S1PR1 in human lung ECs via increased expression of Krüppel-like factor 2 (KLF2), a transcription factor regulator of EC stress responses . Simvastsistance .In our murine RILI model, statin treatment was also found to be protective . In thesWhile there are multiple potential mechanisms underlying RILI protection by simvastatin, evidence suggests that the known effects on sphingolipid signaling are an important factor. In support of this idea, gene expression analyses from these studies identified relevant radiation-induced dysregulation of lung gene expression by statins as well as overrepresented canonical pathways that were both dysregulated by irradiation and differentially effected by simvastatin treatment that included sphingolipid pathway genes.As previously noted, we have identified deubiquitination by UCHL1, a deubiquitinating enzyme, as an important mediator of murine RILI. This finding stems from the identification of GADD45a as a novel candidate gene in pre-clinical models of ALI . SubsequTo further explore the potential role for UCHL1 in RILI, we assessed UCHL1 expression levels from whole lung homogenates of RILI-challenged mice and found significant increases in UCHL1 mRNA and protein relative to controls. In addition, we confirmed that UCHL1 knockdown (siRNA) has a detrimental effect on EC barrier function as measured by transendothelial electrical resistance changes over time in response to both barrier-enhancing and barrier-disruptive agonists. Subsequently, we used a pharmacologic UCHL1 inhibitor, LDN-57444, and confirmed augmented RILI associated with UCHL1 inhibition. Finally, in separate experiments, EC SphK1 levels were inversely affected by UCHL1-silencing and overexpression while inhibition of SphK1 resulted in decreased basal and radiation-induced EC UCHL1 expression .These findings strongly support the idea that SphK1 ubiquitination by UCHL1 is an important determinant of the elaboration of RILI and suggest that strategies aimed at increasing UCHL1 activity could confer RILI protection. To this end, molecules that have been identified to be novel UCHL1 potentiators are particularly intriguing and warrant study in RILI models .There is no question that RILI represents an area of clinical medicine for which current treatments are sorely lacking and for which targeted therapies are non-existent. Accordingly, consideration of previously under-recognized mechanisms that drive RILI pathobiology is warranted in the hope of identifying novel and effective therapies. The sphingolipid pathway certainly merits study in this regard and mounting evidence suggests that direct targeting of sphinglipid signaling with S1P and its analogs could be highly effective for the treatment or prevention of RILI. Moreover, while S1P and several analogs have been studied and shown promise in RILI models, several other analogs have yet to be investigated in this context including CYM-5442, AUY954, and ponesimod, three other S1PR1 agonists –42.Separately, while statin drugs and strategies aimed at augmenting UCHL1 activity are both promising RILI therapies, these are only two examples of possible approaches intended to indirectly augment the EC-protective effects of sphingolipid signaling. Undoubtedly, there are countless other therapies that could leverage this same idea and would warrant their own investigation in this context in an effort to address this unmet medical need. As the role of sphingolipids in RILI pathobiology continues to be defined and becomes more widely recognized, it is virtually certain that related advances will be made that translate directly to changes in patient care practices for those with or at risk for RILI.


Sc) from 120 days post exposure. The accumulation of PrPSc could be intensified by adding a cationic lipid mixture to the infectious brain homogenate at the time of infection. Stable propagation of human prions in a long-term murine glia cell culture represents a new tool for future drug development and for mechanistic studies in the field of human prion biology. In addition, our cell model can reduce the need for bioassays with human prions and thereby contributes to further implementation of the 3R principles aiming at replacement, reduction and refinement of animal experiments.There are various existing cell models for the propagation of animal prions. However, in vitro propagation of human prions has been a long-standing challenge. This study presents the establishment of a long-term primary murine glia culture expressing the human prion protein homozygous for methionine at codon 129, which allows in vitro propagation of Creutzfeldt–Jakob disease (CJD) prions (variant CJD (vCJD) and sporadic CJD (sCJD) type MM2). Prion propagation could be detected by Western blotting of pathological proteinase K-resistant prion protein (PrP C, its TSE-associated pathological isoform is usually designated as PrPSc [TSE [C into its aggregated isoform PrPSc can be triggered spontaneously, genetically or by infection with prions.Prion diseases are a group of progressive and always fatal neurodegenerative disorders also known as transmissible spongiform encephalopathies (TSEs). They include Creutzfeldt–Jakob disease (CJD) and its variant form (vCJD) in humans, scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease (CWD) in cervids. Clinical manifestation of TSEs is characterised by a variety of cognitive and moto-sensory symptoms including dementia, cognitive decline, impaired speech, incoordination, etc. . Accordias PrPSc  or PrPTSPSc [TSE . The conFor the last several decades, prion research has been largely dependent on bioassays and model prions from laboratory animals, which is not least due to the fact that the development of cell models for human prion propagation has proven to be extremely challenging. Thus, animal experiments are often still considered indispensable for drug development, studies on prion biology and the biological detection and titration of TSE agents ,5. HowevSc and possibly mediate its spread from cell to cell via tunnelling nanotubes. The intercellular transfer of aggregated prion protein by astrocytes may have an impact on disease progression [While neurons are the primary target cells for prion replication, it has been shown that other cell types, especially astrocytes and other glia cells, also play important roles in prion biology and are in fact among the earliest sites of prion accumulation. It was further shown that intercellular connections involving astrocytes contain PrPgression . Thus, agression ,8,9,10. gression ,12, invogression ,14.In this report, we present a novel cell assay based on primary glia cell cultures prepared from transgenic mice expressing human PrP homozygous for methionine at codon 129 , which iIsolation and cultivation of primary murine glia from transgenic mice expressing human PrP homozygous for methionine at codon 129 of the prion protein gene (huMM129) were carried out as described previously  with modw/v) as previously described for 263K hamster scrapie prions [Sc , prion replication did not reach detectable levels. Prion propagation was consistently observed in two independently produced huMM129 glia cell preparations and two different samples of both sCJD-MM2 and vCJD homogenate.In cells which were exposed to a 1:100 dilution of infectious brain homogenate  resulted in an apparently stronger PrPSc signal in glia cells challenged with sCJD-MM2 prions at 120 dpe compared to cells which did not receive PTMPTR. The lipid mixture did not have any effect on the extent of vCJD propagation . Cells showed increasing detachment from the culture vessel and displayed an atypical round morphology. Due to the decrease in healthy cells susceptible to prion replication, which was observed from 150 dpe, the signal intensity of PrPSc to non-detectable levels. One of the cell models shown to propagate human prions exploits postmitotic astrocytes [Sc accumulation. Our cell model is based on mixed glia cultures containing mainly astrocytes and, to a smaller degree, microglia, according to findings in similarly prepared cell cultures from hamsters and bank voles [Sc (data not shown). Those cells are highly proliferative and require passaging at least twice a week. PrPSc accumulation might possibly have occurred in these human immortalised astrocytes but was diluted by frequent cell passaging to non-detectable levels.It has previously been reported that (fast) proliferating cells are not able to propagate human prions . This istrocytes , emphasink voles ,16,17. Mnk voles ,19, whicnk voles  to sCJD Sc replication in our cell cultures. Furthermore, we observed a relatively strong cytotoxic effect of sCJD-MM1 prions during the first three days following infection. This initial cytotoxicity possibly resulted in a substantially reduced cell number of viable cells throughout the course of the assay, and therefore the level of PrPSc propagation remained below level of detection. Visual assessment did not disclose a massively reduced cell density in the affected wells, but since we did not evaluate culture properties with viability or cytotoxicity assays, we cannot rule out this possibility.We can only speculate as to why we observed prion propagation for sCJD-MM2 and vCJD but not for sCJD-MM1 prions. One obvious explanation might be that sCJD-MM1 and -MM2 as well as vCJD prions comprise different protein conformations, which differently favour or hinder PrPTM Protein Transfection Reagent  significantly increases the infectivity of RML prions in the scrapie cell assay [Sc signal of sCJD-MM2 prions from 120 dpe compared to non-PTMPTR-treated cells. The variably reduced number of viable cells observed from 180 dpe onwards apparently diminished the enhanced propagation of sCJD-MM2 prions induced by PTMPTR. The effects seen at 120 and 150 dpe may have been caused by coating infectious prions in a positively charged layer of amphipathic molecules, therefore facilitating uptake into the negatively charged cell membrane [TMPTR in hamster glia cultures infected with 263 K scrapie prions  and are therefore confident that the observed effect is not only due to random variation.An explanation as to why we observed enhanced prion propagation for sCJD-MM2 but not for vCJD prions could be that vCJD shows a higher infectivity and attack rate in bioassays compared to sCJD, and that prion propagation in vitro might already occur at a relatively high level, which leaves little room for enhancement  as opposed to sCJD prions.A common transfection method is the reversal of membrane polarity in order to increase membrane permeability by cationic lipids. It has been shown that the cationic lipid Piercell assay . Based omembrane . This waThe simplicity of our cell model in terms of isolation technique and cultivation conditions predestines it to be set up with glia cells of other species, including bank voles, which are considered to be universal acceptors of prion strains . TransgeOnce established for a broader spectrum of human prion strains and possibly further optimised in terms of infection speed and sensitivity, this cell model may substantially facilitate studies on the pathobiology of human prions and drug therapy of CJD.Cells were sourced from neonatal mice, which are null for mouse PrP but express human PrP homozygous for methionine at codon 129 of the prion protein gene . These Euthanasia of huMM129 mice aged 1–3 days for the preparation of brain tissue  was reported to and registered by the local animal protection authority . All animals used in this study were handled under the European directive regarding the protection of animals used for scientific purposes, in strict accordance with the German Animal Welfare Act (Tierschutzgesetz).The work described in this study has been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. Sampling and use of tissue from vCJD and sCJD patients for scientific purposes were carried out with the understanding and written consent of each donor or his or her authorised caregiver. Use of the tissue for experimental studies and scientific reports such as our work was approved for sCJD specimens by the Ethics Committee of the University Medical Center Göttingen (No. 11/11/93) and for vCJD specimens by the East of Scotland Research Ethics Service (No. 16/ES/0084).g for 10 min; the pellet was resuspended in DMEM containing 10% fetal calf serum , 1% GlutaMAX and 100 U/mL penicillin/streptomycin ; and cells were plated on poly-L-lysine-coated  T75 flasks while allocating three brains per flask. Cells were maintained in a 5% CO2 incubator at 37 °C. After two days, cells were washed vigorously four times with PBS and kept in culture for another two to three days. Cells were then split 1:4 and subconfluent cultures were either cryopreserved as described previously [Isolation and cultivation of primary murine glia cells were carried out as previously described  with modeviously  or direc4 cells/well. sCJD-MM1/-MM2 and vCJD 10% homogenates (w/v) were sonicated at 300 W for 30 sec in a microplate horn sonicator (Misonix 3000) prior to cell infection.HuMM129 glia cells were plated in PLL-coated (0.01%) 6-well plates at 3 × 10Sc from the original inoculum.The 10% brain homogenates were diluted 1:10 and 1:100 in DMEM complete and cells were exposed to 10 µL of 1% and 0.1% homogenates in 2 mL culture medium for three days. Cells were then washed once with PBS, to remove residual homogenate from the cultures, and supplemented with fresh medium. Cultures were maintained for 30, 60, 90, 120, 150 and 180 dpe while supplementing them once a week with fresh medium. In order to prove reproducibility, two independent cell isolations of huMM129 glia were used as well as two different aliquots of both sCJD and vCJD homogenates. Samples for Western blot analysis were taken for each time point; one additional sample was taken at 3 dpe in order to indicate the presence of residual PrPTMPTR . To tesSc was performed as previously described [v/v). In total, 10 µL of the cell lysate was digested with 75 µg/mL proteinase K  at 37 °C for 1 h and further denatured in 2× sample loading buffer at 110 °C for 10 min. Ten percent hamster brain homogenate (w/v) of a 263K scrapie-infected animal diluted 1:1000 served as a positive control. SDS-PAGE electrophoresis was performed on BOLT 4–12% Bis-Tris mini protein gels, and proteins were blotted onto PVDF membranes using the iBlot 2 dry blotting system (Thermo Fisher). PVDF membranes were probed with anti-PrP monoclonal antibody 3F4  at 4 °C overnight and anti-mouse IgG alkaline phosphatase-linked secondary antibody . Protein bands were detected with CDP-Star chemiluminescent substrate (Thermo Fisher) for alkaline phosphatase chemiluminescence reaction on Amersham HyperfilmTM ECL films , which were exposed for 40 min.Western blot analysis for the detection of accumulated PrPescribed ,6 with m


To evaluate potential MRI-defined effect modifiers of amoxicillin treatment in patients with chronic low back pain and type 1 or 2 Modic changes (MCs) at the level of a previous lumbar disc herniation (index level).n = 155).In a prospective trial (AIM), 180 patients  were randomised to receive amoxicillin or placebo for 3 months. Primary outcome was the Roland-Morris Disability Questionnaire (RMDQ) score  at 1 year. Mean RMDQ score difference between the groups at 1 year defined the treatment effect; 4 RMDQ points defined the minimal clinically important effect. Predefined baseline MRI features of MCs at the index level(s) were investigated as potential effect modifiers. The predefined primary hypothesis was a better effect of amoxicillin when short tau inversion recovery (STIR) shows more MC-related high signal. To evaluate this hypothesis, we pre-constructed a composite variable with three categories (STIR1/2/3). STIR3 implied MC-related STIR signal increases with volume ≥ 25% and height > 50% of vertebral body and maximum intensity increase ≥ 25% and presence on both sides of the disc. As pre-planned, interaction with treatment was analysed using ANCOVA in the per protocol population (n = 41) and STIR signal volume ≥ 25% alone (n = 45) modified the treatment effect of amoxicillin. As hypothesised, STIR3 patients reported the largest effect .The STIR3 composite group (Predefined subgroups with abundant MC-related index-level oedema on STIR modified the effect of amoxicillin. This finding needs replication and further support.In the primary analysis of the AIM trial, the effect of amoxicillin in patients with chronic low back pain and type 1 or 2 MCs did not reach the predefined cut-off for clinical importance.• In the present MRI subgroup analysis of AIM, predefined subgroups with abundant MC-related oedema on STIR reported an effect of amoxicillin.• This finding requires replication and further support.• The online version contains supplementary material available at 10.1007/s00330-020-07542-w. Cutibacterium acnes bacteria to a previously disrupted, neo-vascularised lumbar disc . The disability at baseline was similar, not worse in the STIR3 group as expected, and cannot explain the difference. These findings make it relevant to examine patients with extensive MC-related oedema on STIR as a separate subgroup in future treatment studies.It remains unclear why the treatment effect (RMDQ difference) at 1 year was smaller in the STIR3 group  than in the prior cohort with type 1 MCs  . BaselinCutibacterium acnes discitis.The STIR3 results were consistent with the hypothesis that some MCs with abundant oedema on STIR might represent low-grade discitis. Importantly, spondylodiscitis was an exclusion criterion and was not suspected on MRI. The STIR3 findings were credible by most of the predefined criteria Table  and the However, replication of our findings is essential. The effect of amoxicillin in the STIR3 group varied greatly Fig.  and was The present findings support the use of STIR to evaluate MCs. They also motivate further studies of MC-related oedema on STIR in relation to possible biological markers of infection that are currently being investigated by our research group. To achieve an optimal classification of MCs for potential clinical use, MC characteristics not studied here, such as diffusion parameters , contrasFurther work is needed to quantify STIR findings. The composite STIR variable was based on both visual assessments and several time-consuming manual measurements. The visually estimated STIR signal volume alone yielded similar results and might be more applicable in a clinical setting. However, precise measurements are preferable in research and may become more feasible with advanced automated techniques , 48. To The strengths of this study include predefined hypotheses, standardised MRI techniques, and MRI ratings by three experienced radiologists . AdditioSubgroup studies of clinical trials often have limited statistical power and generalisability , 53, 54.We evaluated the MRIs at inclusion before defining the subgroups, and the definitions of the MRI subgroups were partly dependent on the MRI data . FurtherPredefined subgroups with chronic LBP, an index level with prior disc herniation, and abundant MC-related index-level oedema on STIR modified the treatment effect of amoxicillin. This finding shows moderate credibility based on published criteria and post hoc analyses and requires replication.ESM 1(DOCX 683 kb)