We studied an accelerated longitudinal cohort of adolescents and young adults  to investigate dynamic reconfigurations in myeloarchitecture. Intracortical profiles were generated using magnetization transfer (MT) data, a myelin-sensitive magnetic resonance imaging contrast. Mixed-effect models of depth specific intracortical profiles demonstrated two separate processes i) overall increases in MT, and ii) flattening of the MT profile related to enhanced signal in mid-to-deeper layers, especially in heteromodal and unimodal association cortices. This development was independent of morphological changes. Enhanced MT in mid-to-deeper layers was found to spatially co-localise specifically with gene expression markers of oligodendrocytes. Interregional covariance analysis revealed that these intracortical changes contributed to a gradual differentiation of higher-order from lower-order systems. Depth-dependent trajectories of intracortical myeloarchitectural development contribute to the maturation of structural hierarchies in the human neocortex, providing a model for adolescent development that bridges microstructural and macroscopic scales of brain organisation. Adolescence is a crucial phase in biological and psychosocial maturation and involves large-scale reconfigurations of brain anatomy . PreviouQuantitative profiling of intracortical properties across cortical depths, and specifically parameterization using central moments , has beeThe present study examined adolescent maturation of cortical architecture and microstructure using an accelerated longitudinal NeuroScience in Psychiatry Network dataset . We tracWe studied the NSPN dataset, an accelerated longitudinal cohort that aggregates multimodal imaging data from 234 adolescents and young adults aged 14–27 . Briefly, we generated cortical surface models based on T1-weighted MRI and co-registered the corresponding MT volumes to these surfaces. We systematically generated equivolumetric intracortical surfaces and sampled MT intensities along matched vertices perpendicular to the cortical mantle to build intracortical MT profiles . Our veri.e., using the average across timepoint 1), the cortex-wide average MT profile shows a non-linear increase in intensity from the superficial layers, adjacent to the pial boundary, towards the deeper layers approaching the white matter boundary, consistent with prior literature .We explored the topology of intracortical MT patterns using microstructure profile covariance (MPC). By correlating depth-wise MT profiles, the MPC procedure estimates inter-regional microstructural similarity and has previously been validated using a combination of post mortem and in vivo data . DiffusiFDR <0.00625, % rate of change: [4.8 14.7] 95% CIs; FDR <0.00625, % rate of change: [−179.2–34.6] 95% CIs; Studying age-related changes in MT moments, we observed that adolescence led to a significant increase in the mean , we conducted enrichment analysis of standard mammalian phenotypes . Cell specific expression analysis . This is expected since those areas of high expressing myelin genes are most vulnerable  illustrated a sensory-fugal gradient, explaining 33% variance (DEV) with the baseline MPC gradient  within the youngest (<16 years) and oldest (>24 years) age strata. Similar results were observed using alternative age windows. Heteromodal and unimodal cortex, occupying central regions of the gradient, were drawn outwards towards one of the gradient anchors in older individuals . Together, these findings demonstrate how depth-dependent myelination during adolescence reshapes myeloarchitecture and underpins macro-scale reorganisation of cortical networks.Adopting the MPC framework, we subsequently assessed inter-regional coordination of myeloarchitectural development. MPC networks were constructed for each participant, and we modelled age-related changes in microstructural similarity between all node pairs . At the variance . On one ividuals , formingividuals . Specifiividuals . By relaOur longitudinal analyses revealed marked alterations in intracortical myeloarchitecture during adolescence, which was accompanied by large-scale reorganisation of microstructural gradients. Changes to intracortical myelin can be generally characterised by two developmental processes, one involving overall increases in mean MT and one involving preferential MT increases in mid-to-deeper cortical layers. Both processes particularly affected unimodal and heteromodal association cortices that is areas between low-level sensory and motor areas on the one end of the cortical hierarchy and high-level transmodal and limbic regions on the other end. A series of complementary explorations, based on alternative neuroimaging features known to undergo large-scale changes in adolescence , demonstInspired by classical histological studies operating on 2D sections , our worTracking longitudinal change in intracortical MT profiles, we found concomitant but independent developmental patterns in the trajectory of mean and skewness. Both sets of age-related changes were strongly and preferentially located in heteromodal and unimodal association cortex; yet, they only partially overlapped, a finding also supported by gene expression analysis. Only skewness changes, but not mean MT changes, were spatially associated with oligodendrocyte gene expression patterns. Additional analyses indicated that skewness effects occurred rather independently of MRI-based measures of cortical interface blurring and variations in cortical thickness, supporting specificity to intracortical microstructure. Our findings may align with several mechanisms of experience-dependent myelination . IncreasAdaptation of a recent microstructure profile covariance analysis framework to longitudinal MT data illustrated the macroscale impact of microstructural changes. Specifically, decreases in MT profile skewness pushed regions to a more sensory-like architecture (which was coupled with high mean MT). In contrast, increases in skewness pushed regions towards a paralimbic-like MT profile. These regions also underwent protracted increases in mean MT, but still did not reach the high levels observed in sensory regions. These two regionally distinct developmental patterns within the association cortex promoted a more bimodal distribution of myeloarchitectural types across the cortex. Similar properties of modular segregation in tractography-based networks have been shown to mediate age-related improvements in executive function throughout adolescence , reinforOur work suggests that myelination during adolescence is unlikely to be a question of simply more or less. Instead, our longitudinal findings show that the type of change is topologically divergent when we take depth into consideration. Expanding upon previous evidence of adolescent increases in overall mean intracortical myelin content, our findings demonstrate a relative preferential specific accumulation of myelin towards mid-to-deeper infragranular layers mainly in association cortices. As our analyses have shown, these findings are not explainable by changes in overall cortical morphology during adolescence, but instead likely reflect architectural changes associated with oligodendrocyte related processes. The coordinated change patterns strengthen the notion that the forces of adolescent development further widen the existing axis of macroscale cortical organization, driving association cortex either closer towards sensory or limbic systems. Thus, our findings illustrate how cell-type and layer specific microstructural changes assessed in the direction of cortical columns contribute to the maturation of macroscale cortical organisation and suggest adolescent calibration of structural hierarchical gradients.The present study included a subset of individuals from the NeuroScience in Psychiatry Network (NSPN) study . For a vT1w and MT imaging were acquired as part of the quantitative multiparameter mapping (MPM) sequence  on threeAll raw images were visually inspected by experienced researchers and 11 participants were excluded due to excessive motion artefacts. Surface reconstructions were visually inspected and manually edited for all scans, up to 10 times . At eachi.e.,: myelin is more white), obtained intensity values per pixel by reading the image into MATLAB (v2017b), then generated a region-specific intensity profile by averaging values row-wise. Finally, we calculated the MT moments for each region and contrasted these values with baseline group-average MT moments for matched regions. Additionally, we contrasted baseline group-average MT moments across levels of laminar differentiation , skewness, and kurtosis . We focuntiation  and cytontiation  to deterntiation , we perfntiation . All span represents the node. The addition of person-specific random intercepts significantly improves model fit in accelerated longitudinal designs. LMEs were performed on each node and t-statistics were projected onto the cortical surface. Additionally, we examined the relationship between level of laminar differentiation and adolescent development of MT moments. To assess the relative over- or under-representation of significant regions per laminar class we generated 10,000 spin permutations of the t-maps, which controls for spatial contiguity and hemispheric symmetry across permutations  correction for multiple comparisons  using SurfStat  for Matlutations . Specifiparisons  to creatZ-score modification of Fisher’s exact test and FDR correction. It should be noted that the AIBS data are based on adult post-mortem and that the developmental associations are indirect associations between the enriched gene-set from spatial overlap analysis and a different developmental gene-expression dataset. Specifically, the t-statistic maps of age-related changes in the moments were spatially correlated with gene expression maps from the AIBS dataset using Neurovault (https://github.com/NeuroVault/NeuroVault/tree/master/ahba_docker). The genes derived from this spatial analysis were subsequently compared against developmental expression profiles from the BrainSpan dataset (http://www.brainspan.org/) using the CSEA developmental expression tool developed by the Dougherty lab (http://genetics.wustl.edu/jdlab/csea-tool-2/).We measured the spatial overlap between our baseline maps of MT moments , depict the similarity of nodal patterns of MPC change. Similar to previous analyses  bin-wise Cohen’s d effect size change of gradient values, (iii) node-wise difference in MPC to the gradient anchors, that is the extreme bins, and (iv) bin-wise difference in MT moment values. To reconcile macroscale changes in the MPC gradients to microstructural alterations, we performed a spatial Pearson correlation between unthresholded t-statistic maps of age-related changes in MT mean and skewness .Preprocessed microstructure profiles are available via GitHub: The repository also includes Matlab and R scripts to reproduce the primary analyses and figures. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.Acceptance Summary:This novel and important study capitalizes upon a large sample of youths scanned twice with magnetization transfer imaging, in order to map developmental profiles of cortical myelin. The authors use this approach to demonstrate that the human cortex undergoes dissociable, depth-specific changes in cortical myeloarchitecture during development. Critically, greater imaging markers of myelin were spatially associated with gene expression markers of oligodentrocytes, and align with a gradient ranging from primary sensory to higher-order association cortex. Together, this approach provides a new understanding of how cortical myelin develops during the critical period of adolescence. Furthermore, this data has numerous implications for understanding both healthy brain maturation and abnormal brain development associated with neuropsychiatric syndromes.Decision letter after peer review:eLife. Your article has been reviewed by Joshua Gold as the Senior Editor, a Reviewing Editor, and two reviewers. The following individuals involved in review of your submission have agreed to reveal their identity: Kate Mills (Reviewer #1); Ben Fulcher (Reviewer #2).Thank you for submitting your article "Shifts in myeloarchitecture characterise adolescent development of cortical gradients" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This is a study investigating intracortical microstructure in a sample of 223 individuals scanned twice between ages 14-27 years. Authors estimated myelin content in the cortex of magnetic resonance images using magnetization transfer, and by using central moments to test the hypothesis that mean and skewness of the magnetization transfer profiles would show different developmental trajectories and relate to different gene expression patterns. The study is comprehensive and the methods are sound. Writing and presentation are excellent, and code for reproducing the analyses is made available (and processed data where possible), further enhancing the impact of this work. Nonetheless, reviewers felt that several aspects of the work could be strengthened on revision.Essential revisions:1) NSPN sample. More details about the full sample (the full NSPN sample) compared to the included sample for this study would be useful. A visualization of the sampling design would be helpful.2) Quality control. More details regarding the exclusion criteria and brain image quality control procedures are needed. What was the procedure for inspection, criteria used to determine exclusion, and number of scans excluded? Were both the raw images and processed images inspected for quality? Please describe the extent of manual intervention of processed images, including the protocol and number of scans that were successfully processed post-intervention and included in analyses.myelin processes during adolescence").3) AIBS. Further details regarding the processing of the gene expression maps is necessary . Furthermore, authors should clearly state that AIBS data are based on adult postmortem oligodendrocyte density. As such, it seems warranted to temper their claims  MT and myelin. The authors should clarify how sensitive is MT to myelin. The strength of this link is crucial to a lot of the interpretation and biological novelty of the results. MT (like T1w:T2w) may be sensitive not just to myelin, but also potentially other biological variables. Some evidence/discussion of this, and the extent to which the reader can safely make the association to myelin content would be helpful in the introduction. Furthermore, many features are different between the myelin stains and MT profiles . Some brief discussion of the differences would be helpful. Finally, it would be useful to clarify if to the author's knowledge this is the first time anyone has matched MT depth profiles to a histological measurement (or discuss relevant literature of past efforts).5) MT skewness and relative layer thickness. MT skewness may depend on the relative thickness of the different layers. It would be useful to clarify how much results explain the data beyond a simple cytoarchitectural analysis  Furthermore, it would be useful to clarify if every node is squashed to the same absolute depth? This could impact interpretation: two areas with an identical ratio of myelination in deep relative to superficial layers would have different skewnesses if their middle layer was made thicker or thinner (thicker middle layer could push out the skewness). If this is the case, MT skewness could not simply be interpreted as a "ratio of myelination in deeper compared to more superficial layers"; it would more represent a measure of the preferential distribution of MT closer to the white matter boundary. Clarification is needed.6) Statistical testing. Every time a statistical analysis is performed , it should be clearly stated to the reader what it is. Non-normal distributions seem common in this data; Spearman's correlations may be appropriate. Furthermore, it appears that the spin-test based permutations are only used for some analyses but not others . Were spin tests used for earlier spatial association analyses? If not, can this be justified?7) Utility of low-dimensional embedding. Regarding this statement: "Nodes closer in this embedding space increase in microstructural similarity during adolescence, whereas distant nodes decouple", it is unclear why a low-dimensional embedding is useful a direct measurement of change is available. Couldn't you more easily do an analysis directly  What is gained from this harder-to-interpret nonlinear embedding that helps test/interpret your hypotheses? Perhaps more importantly, it is unclear if this statement is supported by the data: Wouldn't the embedding put nodes close in the space that have similar "patterns" of age-related MPC change with other network nodes? In the plot of the behavior of G1_dev, it does not display the claimed behavior  – instead, it seems like the largest values of G1_dev already have ~maximal G1_MPC, and thus barely change at all between the <16 and >24 groups. Clarification is warranted.8) Biological interpretation. In the discussion, the biological interpretation of the results at times is rather thin. If MT is a strong myelin marker, then why would myeloarchitecture be reorganizing like this ? Axonal myelination improves transmission speeds and prevents the formation of new synaptic connections. Could you speculate on how this might alter behavior relevant to the longitudinal changes analyzed? Relevant papers to consider include:*Micheva et al., (2016).*Braitenberg, (1962).*Huntenburg et al., (2017). Summary:This is a study investigating intracortical microstructure in a sample of 223 individuals scanned twice between ages 14-27 years. Authors estimated myelin content in the cortex of magnetic resonance images using magnetization transfer, and by using central moments to test the hypothesis that mean and skewness of the magnetization transfer profiles would show different developmental trajectories and relate to different gene expression patterns. The study is comprehensive and the methods are sound. Writing and presentation are excellent, and code for reproducing the analyses is made available (and processed data where possible), further enhancing the impact of this work. Nonetheless, reviewers felt that several aspects of the work could be strengthened on revision.We thank the reviewers and Editor for recognising the quality and impact of our work. We are grateful to the reviewers for their constructive comments, which we feel have improved the manuscript. We have addressed all comments point-by-point.Essential revisions:1) NSPN sample. More details about the full sample (the full NSPN sample) compared to the included sample for this study would be useful. A visualization of the sampling design would be helpful.We thank the reviewers for this suggestion and have provided greater characterisation of our cohort in the Materials and methods section. A visualization is provided in the new Appendix—figure 6.“The present study included a subset of individuals from the NeuroScience in Psychiatry Network (NSPN) study . […] Secondary cohort data were eligible for inclusion in this study if T1w and MT data were available at two timepoints, resulting in N=234 healthy adolescents. Following additional quality control on raw images, surface reconstructions and MT profiles, detailed below, the evaluable sample included N=180 individuals .”2) Quality control. More details regarding the exclusion criteria and brain image quality control procedures are needed. What was the procedure for inspection, criteria used to determine exclusion, and number of scans excluded? Were both the raw images and processed images inspected for quality? Please describe the extent of manual intervention of processed images, including the protocol and number of scans that were successfully processed post-intervention and included in analyses.To clarify, quality control and exclusion criteria were conducted at three stages: (a) on the raw images, (b) at the level surface reconstructions, and c) with respect to the MT profiles. As suggested, we have now provided more specifics in the revised Materials and methods section:“All raw images were visually inspected by experienced researchers and 11 participants were excluded due to excessive motion artefacts. Surface reconstructions were visually inspected and manually edited for all scans, up to 10 times . At each iteration, control points and grey/white matter edits were included, and the surface reconstruction was repeated. 10 participants were excluded due to poor surface reconstructions.”“Prior to statistical assessment, we identified and removed outlier individuals (n=33). Outliers were defined as individuals with >1% nodes that deviated from the age-stratified median of nodal mean MT by >3 interquartile ranges.”3) AIBS. Further details regarding the processing of the gene expression maps is necessary . Furthermore, authors should clearly state that AIBS data are based on adult postmortem oligodendrocyte density. As such, it seems warranted to temper their claims .We thank the reviewers for this important comment and are happy to provide further clarifications. The present analysis relied on the Neurovault implementation of AIBS gene expression processing. More details regarding the automated gene decoding are provided in the Materials and methods section:“We measured the spatial overlap between our baseline maps of MT moments  and t-statistics  and maps of post mortem gene expression from the Allen Institute for Brain Sciences (AIBS). […] Resultant gene associations p-values were corrected at an FDR level of 0.05 across included genes and only genes passing correction were included.”As suggested, we tempered claims regarding the adult post-mortem nature of gene expression data in the updated Results section:“Both analyses thus confirmed a spatial overlap of our findings from NSPN with genes associated with myelin processes that are also enriched during adolescence. […] While there was thus evidence for an adolescent developmental signal, results are nevertheless indirect also given that glial and oligodendrocyte associated genes may undergo expression changes during other periods of the lifespan .”We also added the following caveat to the Materials and methods section:“It should be noted that the AIBS data is based on adult post-mortem and that the developmental associations are indirect associations between the enriched gene-set from spatial overlap analysis and a different developmental gene-expression dataset. Specifically, the t-statistic maps of age-related changes in the moments were spatially correlated with gene expression maps from the AIBS dataset using Neurovault (https://github.com/NeuroVault/NeuroVault/tree/master/ahba_docker). The genes derived from this spatial analysis were subsequently compared against developmental expression profiles from the BrainSpan dataset (http://www.brainspan.org/) using the CSEA developmental expression tool developed by the Dougherty lab (http://genetics.wustl.edu/jdlab/csea-tool-2/).”4) MT and myelin. The authors should clarify how sensitive is MT to myelin. The strength of this link is crucial to a lot of the interpretation and biological novelty of the results. MT (like T1w:T2w) may be sensitive not just to myelin, but also potentially other biological variables. Some evidence/discussion of this, and the extent to which the reader can safely make the association to myelin content would be helpful in the introduction. Furthermore, many features are different between the myelin stains and MT profiles . Some brief discussion of the differences would be helpful. Finally, it would be useful to clarify if to the author's knowledge this is the first time anyone has matched MT depth profiles to a histological measurement (or discuss relevant literature of past efforts).The updated manuscript elaborated more on the relationship between MT, myelin, and the link between MT profiles and intracortical architecture. As suggested, we also extended our Discussion section on our novel comparison of MT profiles with myelin sections, specifically highlighting that differences in resolution and specificity to myelin produce differences in the profiles.Updates were made to the Introduction, Results section and Materials and methods section:“Several recent neuroimaging studies assessed intracortical microstructure in adolescence. One promising imaging technique is magnetisation transfer (MT), an MRI acquisition sequence that is sensitive to how water molecules interact with macromolecules in the brain, notably myelin . Although techniques such as MT cannot be equated with cortical myelin content per se , the MT parameter is dominated by myelin-related molecules making this technique a viable in vivo proxy for the contrast seen histologically in myelin basic protein . A post mortem study in patients with multiple sclerosis has also shown that MT measures scale with demyelination and remyelination, suggesting dependence of this measure on myelin content .”“Although post mortem myelin-stained sections and in vivo MT profiles differ in terms of resolution and specificity to myelin, we observed similar variations in mean and skewness of profiles across levels of laminar differentiation , supporting the extension of profile analysis from histology to in vivo MT imaging.”“The MT saturation parameter decouples the MT signal from the longitudinal relaxation rate, making it a semi-quantitative measure that is robust to relaxation times and field inhomogeneities .”“To further understand architectural and cellular underpinnings of in vivo MT profiles, we assessed the similarity of the MT-derived profiles with intracortical myelin profiles computed from previously published post mortem myelin sections. While there are clear differences in the resolution and specificity to myelin between the histological staining and our in vivo proxy data, findings were overall supportive of a close association between in vivo MT profiles and those derived from myelin stained sections. This line of evidence supports the specificity of the MT features to intracortical myeloarchitecture and motivates efforts to increase the availability of post mortem histological measures for more detailed cross-validation studies of putative MRI markers of myelin.”5) MT skewness and relative layer thickness. MT skewness may depend on the relative thickness of the different layers. It would be useful to clarify how much results explain the data beyond a simple cytoarchitectural analysis  Furthermore, it would be useful to clarify if every node is squashed to the same absolute depth? This could impact interpretation: two areas with an identical ratio of myelination in deep relative to superficial layers would have different skewnesses if their middle layer was made thicker or thinner (thicker middle layer could push out the skewness). If this is the case, MT skewness could not simply be interpreted as a "ratio of myelination in deeper compared to more superficial layers"; it would more represent a measure of the preferential distribution of MT closer to the white matter boundary. Clarification is needed.We thank the reviewers for this interesting question. We have carried out several precautions and additional analyses to dispel an interaction of thickness with MT profile results. First, please note that our MT sampling approach leveraged equivolumetric transformations for intracortical surface constructions , which accounted for variations in the laminar thickness with respect to curvature. Secondly, we have regressed out overall cortical thickness from the MT findings in Appendix-figure 2 and observed no major changes to the spatial pattern of MT moments or age related changes in MT moments when including thickness in the regression model. The revised paper furthermore tested whether laminar thickness had an effect on MT skewness. Specifically, we extracted laminar thickness from a recent segmentation of cortical layers in a post mortem volumetric reconstruction of a human brain . We found that laminar thicknesses did not exhibit systematic relationships with skewness (-0.17<r<0.02). We have updated the Results section and added these findings into a new Appendix-figure 5:“Furthermore, spatial correlation analysis suggested that MT moments were relatively independent of regional variations in laminar thickness derived from a post mortem volumetric reconstruction of a Merker-stained human brain  .”6) Statistical testing. Every time a statistical analysis is performed , it should be clearly stated to the reader what it is. Non-normal distributions seem common in this data; Spearman's correlations may be appropriate. Furthermore, it appears that the spin-test based permutations are only used for some analyses but not others . Were spin tests used for earlier spatial association analyses? If not, can this be justified?We thank the reviewers for this suggestion. In the updated manuscript, all spatial correlation analyses utilized Spearman correlations and corresponding p-values were determined via spin tests . We have updated Figure 1 and Figure 4 to reflect the slight changes in statistics. The Results section and Materials and methods section have also been amended as follows:spin<0.001; Figure 1C).”“At a whole cortex-level, mean MT was highest in idiotypic cortex and decreased with less laminar differentiation, while skewness exhibited an opposite pattern  and weakly with mean MT .”“This sensory-fugal gradient reflects systematic variations in the MT profiles; it was strongly correlated with MT profile skewness . Conversely, restricted age-related skewness changes were associated with paralimbic anchor coupling .”“By relating age-related shifts in MPC to anchors with age-related changes in MT moments, we found that greater decreases in MT skewness were related to sensory anchor coupling . All spatial correlations were estimated and tested in the same way throughout the study.”7) Utility of low-dimensional embedding. Regarding this statement: "Nodes closer in this embedding space increase in microstructural similarity during adolescence, whereas distant nodes decouple", it is unclear why a low-dimensional embedding is useful a direct measurement of change is available. Couldn't you more easily do an analysis directly  What is gained from this harder-to-interpret nonlinear embedding that helps test/interpret your hypotheses? Perhaps more importantly, it is unclear if this statement is supported by the data: Wouldn't the embedding put nodes close in the space that have similar "patterns" of age-related MPC change with other network nodes? In the plot of the behavior of G1_dev, it does not display the claimed behavior  – instead, it seems like the largest values of G1_dev already have ~maximal G1_MPC, and thus barely change at all between the <16 and >24 groups. Clarification is warranted.We agree with the reviewers’ recommendation that the G1_dev would be better described as “Nodes closer in this embedding space have more similar patterns of age-related change in MPC, whereas distant nodes undergo dissimilar development” (updated in the Results section). It follows from this definition that positioning of G1_dev reflects the principal axis of variance in developmental changes, but that does not necessarily correspond to changes in the values of G1_MPC.FDR<0.025), but they were not clearly circumscribed to a set of brain regions or level of laminar differentiation , therefore the low-dimensional embedding was helpful to visualise and interpret the patterns of change. In fact, the embedding approach led to the discovery that adolescent development expanded the existing sensory-fugal gradient of microstructural differentiation, and this would not have been easily observable when using the high-dimensional representations.Low-dimensional embedding was used to synoptically characterise coordinated age-related changes in MPC. We noted many significant age-related changes in MPC (1564 increases vs 534 decreases at q8) Biological interpretation. In the discussion, the biological interpretation of the results at times is rather thin. If MT is a strong myelin marker, then why would myeloarchitecture be reorganizing like this ? Axonal myelination improves transmission speeds and prevents the formation of new synaptic connections. Could you speculate on how this might alter behavior relevant to the longitudinal changes analyzed? Relevant papers to consider include:*Micheva et al., (2016).*Braitenberg, (1962).*Huntenburg et al., (2017).We have expanded the biological interpretation of our findings and provided further speculation on potential implications for behaviour, also citing the suggested papers.:“Both sets of age-related changes were strongly and preferentially located in heteromodal and unimodal association cortex; yet, they only partially overlapped, a finding also supported by gene expression analysis. […] The architectural changes observed in the present cohort may represent a microstructural substrate that could determine overall cognitive and social capacities or susceptibility to a range of neuropsychiatric and neurological conditions occurring in late childhood and adolescence.”


The ability to interpret transcranial magnetic stimulation (TMS)-evoked electroencephalography (EEG) potentials (TEPs) is limited by artifacts, such as auditory evoked responses produced by discharge of the TMS coil. TEPs generated from direct cortical stimulation should vary in their topographical activity pattern according to stimulation site and differ from responses to sham stimulation. Responses that do not show these effects are likely to be artifactual. In 20 healthy volunteers, we delivered active and sham TMS to the right prefrontal, left primary motor, and left posterior parietal cortex and compared the waveform similarity of TEPs between stimulation sites and active and sham TMS using a cosine similarity-based analysis method. We identified epochs after the stimulus when the spatial pattern of TMS-evoked activation showed greater than random similarity between stimulation sites and sham vs. active TMS, indicating the presence of a dominant artifact. To do this, we binarized the derivatives of the TEPs recorded from 30 EEG channels and calculated cosine similarity between conditions at each time point with millisecond resolution. Only TEP components occurring before approximately 80 ms differed across stimulation sites and between active and sham, indicating site and condition-specific responses. We therefore conclude that, in the absence of noise masking or other measures to decrease neural artifact, TEP components before about 80 ms can be safely interpreted as stimulation location-specific responses to TMS, but components beyond this latency should be interpreted with caution due to high similarity in their topographical activity pattern. Transcranial magnetic stimulation (TMS)-evoked EEG potentials (TEPs) are a rich source of neurophysiological data and have been used to study a variety of phenomena, including consciousness ,2, memorRecent work has demonstrated that TEPs differ in waveform shape across recording and stimulation sites , and areDue to the likely impossibility of completely removing brain-generated artifacts, such as auditory and somatosensory responses, it is important to identify which epochs of the TEP contain such artifacts and to interpret signals from those epochs with caution or avoid them altogether. Previous studies have shown that a late TEP component, the N100, is similar in scalp distribution irrespective of stimulation site and between active and sham stimulation ,20. The Alternatively, by calculating the instantaneous cosine similarity between waveforms, similarity can be assessed at each sampled time point. Cosine similarity compares evoked responses across multiple channels and can be used to measure similarity in the shapes of responses across a set of electrodes . In ordeWe used the cosine similarity method to measure similarity in TEPs resulting from active and sham TMS delivered to three different stimulation sites, with the hypothesis that TEPs generated by stimulation at different sites should produce different topographic distributions of activity when the recording is not dominated by non-site-specific activity, i.e., artifact. The objective of this study was to demonstrate the ability of the cosine similarity method to detect artifact.Twenty healthy individuals  provided informed consent to participate in the study, which was approved by the NIH Combined Central Nervous System Institutional Review Board. All experiments were performed in accordance with the Declaration of Helsinki.1-weighted scans were obtained from each participant using either a GE 3T HDx scanner  or a Siemens 3T Skyra scanner . Predetermined TMS target locations for the right dorsolateral prefrontal cortex  [MPRAGE T42 46 4) . The tar42 46 4)   > 50 uV on ten consecutive trials. Resting motor threshold (RMT) was calculated using an automatic threshold-tracking algorithm  . StimulaWe delivered 150 active and 50 sham TMS pulses at each stimulation site. The order of sites was counterbalanced between participants. Pulses were delivered every five seconds with a 15% jitter to reduce any effects of pulse anticipation. EEG signals were sampled at 5000 Hz (DC filtered at 1000 Hz) from 30 recording scalp electrodes in a 10–20 arrangement using a TMS-compatible EEG system . Electrode impedances were maintained at < 10 kΩ. The goal of sham stimulation was to reduce cortical stimulation without eliminating the auditory or somatosensory artifacts. This was achieved by placing a 3 cm cardboard spacer between the coil and scalp. For each location, active TMS was performed first, followed by sham. Participants had foam earplugs placed during all TMS procedures.We preprocessed the data with a standard analysis pipeline  extension for EEGLAB  running We used a modified cosine similarity method  to measuSt is cosine similarity at time t, i,tA and i,tB are the binarized derivatives for each condition at channel i and time t, and n is the total number of channels. The process was repeated 1000 times for each participant and each comparison, using randomly-permuted trial averages. The within-participant mean of the averages was then calculated as the measure of similarity between stimulation sites (between-site similarity), stimulation types (between-stimulation similarity) and within-condition repeatability.For each condition, we created an average waveform from 50 randomly-selected trials from each participant. Since only 50 sham trials were delivered, we used 50 trials to equalize the number of trials between comparisons (active vs. sham and between-site active stimulation). Non-overlapping trials were used for within-condition repeatability comparisons. We obtained the derivatives of the preprocessed and averaged TEPs at every time point for each channel by subtracting the amplitude of each timepoint from the amplitude of preceding one . The derFor the between-condition similarity and within-condition repeatability analyses (see above), we used cluster-based permutation testing  to perfop-value was calculated for each time point by comparing the distributions of the participants’ baseline and response epochs. Clusters of significant time-points were identified by summing the p-values of all contiguous time points that reached a threshold of 0.05. We controlled for multiple comparisons by comparing each of the cluster sizes to a distribution of 1000 p-value clusters derived from randomly permuted epoch comparisons and retaining only original clusters > 95% of the random cluster distribution. Each random cluster was created by comparing two randomly-sampled distributions of twenty epochs each from the combined pool of the baseline and response epochs and selecting the size of the largest cluster by summed p-value, as above.We performed statistical comparisons to reveal significant differences between baseline and post-TMS within-condition repeatability and between-condition similarity. For each comparison, each participant’s average within-condition repeatability and between-condition similarity curves were segmented into baseline (1500–500 ms before TMS) and response (15–1015 ms after TMS) epochs. A Active TMS produced significant cortical activation at the M1, PPC, and DLPFC sites, with maximum group-level peak-to-peak amplitudes across the scalp of 13.9 μV, 12.9 μV, and 23.0 μV, respectively . Sham TMQualitatively, TEPs differed spatially between 15 ms (end of interpolation) and 60 ms . BetweenThe within-condition repeatability of TEP waveforms was maximal immediately following the pulse for all stimulation sites and decayed to pre-stimulus values by 346 ± 26 ms after the TMS pulse . SignifiTwo epochs of significantly increased similarity from baseline were common to all six between-site and between-stimulation comparisons . The firWe compared similarity in the spatial distributions of TEPs from active and sham TMS, from active stimulation at different sites, and between samples of trials from the same sites using the cosine similarity method. These comparisons enabled identification of epochs during which TEP activation patterns were similar between sites and conditions, indicating the presence of neural artifact, likely due to auditory and/or somatosensory activation. Our results are consistent with previous findings , in thatWe found that the within-condition repeatability of whole-brain TEP patterns decreased over time , with a The objective of this study was to use cosine similarity to distinguish between responses to cortical stimulation and neurally generated sensory artifacts, such as auditory and somatosensory responses, in the TEP. Therefore, we did not attempt to mask these stimuli. We acknowledge that our results may apply only when auditory and somatosensory artifact is present, although Conde et al.  found unThe cosine similarity approach as applied here has limitations. First, it is based on temporal similarity across EEG channels. Non-source localized EEG data suffers from poor spatial resolution due to volume conduction. We chose not to source-localize our data because we only used 30 active channels, which is likely insufficient for solving the inverse problem . HoweverCosine similarity is a simple and effective tool for identifying TEP epochs when neural artifact is likely to be present. Although artifact may be present throughout the TEP response, our results show that early components best reflect site and condition-specific responses to cortical stimulation. In contrast, later responses become progressively more similar between stimulation conditions, suggesting that these components predominately reflect neural artifacts. We suggest that only TEP components occurring prior to 80 ms are safely interpretable as condition and site-specific responses to cortical stimulation when noise masking is not used. In addition, TEP waveform reproducibility decays monotonically with increasing time after the TMS pulse. While later components may contain valuable physiological information, we suggest caution in in their interpretation and warn against their attribution to direct cortical stimulation by the TMS pulse. Auditory and somatosensory stimulation controls and/or further refinements in signal processing may be necessary to extract true late responses to cortical stimulation.


PurposeA new dual-layer multi-leaf collimator (MLC) system with several improved characteristics was introduced with the Varian Halcyon™ treatment platform. This study evaluated this new MLC’s impact on head and neck plan quality and delivery efficiency.MethodsNine patients were retrospectively studied with Institutional Review Board (IRB) approval. To compare plan quality between the Halcyon dual-layer MLC and Truebeam® MLC, all patients were replanned with the same prescription and target coverage following the institutional clinical protocol for both platforms and using both intensity modulated radiation therapy (IMRT) or volumetrically modulated arc therapy (VMAT) techniques. Organs-at-risk (OAR) dose-volume histogram (DVH) statistics were compared along with total plan monitor units (MU). To evaluate delivery efficiency, actual beam-on time for five patients’ plans were recorded and compared. To evaluate the impact of MLC performance parameters on plan quality, virtual MLC models were generated by matching Truebeam MLC’s parameters to those of the Halcyon dual-layer MLC both individually and combined. OAR doses were then compared between these virtual MLCs, the Truebeam MLC, and the actual Halcyon MLC.ResultsOverall the Halcyon dual-layer MLC provided similar plan quality compared to Truebeam MLC for VMAT plans, and improved sparing for majority of the OARs when using IMRT. Paired comparison showed median dose differences in mean doses to the parotids, cochlea, esophagus, and larynx ranged from -0.83 Gy to 0.37 Gy for VMAT, and from -4.79 Gy to -0.04 Gy for IMRT, with negative values indicating improved performance by Halcyon. Despite a slight increase in plan MU, the Halcyon reduced the total beam-on time by 42.8 ± 8.5%. Virtual MLC simulations demonstrated that matching MLC transmission accounted for nearly half of the total dose difference between Halcyon and Truebeam IMRT plans.ConclusionWhen compared to the Truebeam, the Halcyon’s dual-layer MLC achieved similar plan quality using VMAT, and improved OAR sparing using IMRT, while providing nearly twice as fast treatment delivery. Reduction in MLC transmission is the dominating factor contributing to dosimetric differences in OAR sparing. Radiation therapy plays a key role in managing head and neck cancer. Due to the complexity of the shape and configuration of tumor and lymph nodes in the target region, and close proximity of organs-at-risk (OARs), intensity-modulated radiation therapy (IMRT) or volumetrically modulated arc therapy (VMAT) has been widely used in treatment planning for head neck patients.The delivery of IMRT/VMAT treatment plans relies heavily on the multi-leaf collimator (MLC). Therefore, it is expected that MLC parameters such as inter-leaf leakage, leaf transmission, dosimetric leaf gap (DLG), leaf width, leaf over-travel limits, and leaf speed, play important roles in impacting the quality of the treatment plan. Previous studies have heavily investigated the impact of MLC leaf width on the quality of IMRT plans. Burmeister et al.  and Wu eRecently a new straight-through jawless treatment delivery system was introduced by Varian Medical Systems  trademarked Halcyon™. In this system, a new dual-layer MLC design was introduced to fulfill jawless configuration while simultaneously providing sufficient beam attenuation and shape modulation. The new MLC design, referred to here as “dual-layer MLC,” features a dual-layer of stacked and staggered MLC leaves with 1 cm leaf width in each layer when projected to the isocenter plane, which is at 100 cm source-to-axis distance (SAD). This MLC system also features faster speed than Varian’s current mainstream MLC system used in the Truebeam® accelerator product lines, the Millennium 120, referred to here as TB MLC, providing 5.0 cm/s movement at the isocenter plane compared to 2.5 cm/s for the Millennium 120. The Halcyon dual-layer MLC can also achieve full-field modulation with 100% leave over-travel and interdigitation. The Halcyon linac system offers only the six megavoltage (6 MV) flattening-filter-free (6FFF) mode for treatment delivery. The omission of flattening filter increases the beam output and has a lower average energy than 6 MV beams produced with the same maximal energy but flattened with a filter (6X). This lower average energy, combined with the improvement in the MLC design, provided a significant improvement in the leakage and transmission properties of the dual-layer MLC compared to Millennium 120 MLC: 0.7% per layer leaf transmission for the dual-layer MLC compared to around 1.5% leaf transmission for Millennium 120 used in the Truebeam linac. Dosimetric leaf gap for this dual-layer MLC was changed to 0.1 mm in the Halcyon beam model. At the time of this study, the dual-layer MLC is only able to provide modulation using the distal layer of MLC, i.e., the layer away from the radiation source and closer to the patient, while the proximal MLC layer follows the opening of distal layer MLC, providing additional shielding essentially similar to backup jaws but for each individual interleaf region. Therefore, in this study the effective leaf width for all Halcyon plans is 1.0 cm, compared to 0.5 cm for the regular Truebeam Millennium 120 MLC design. In the recently released Halcyon 2.0 both layers of MLC participate in active fluence modulation, for which results were still being generated and not included in this study.Since the dual-layer MLC has improved transmission, speed, over-travel, and DLG, but twice as large leaf width, it is important to understand the impact to plan quality and delivery efficiency when compared to the regular Truebeam Millennium 120 MLC design. In the first part of this study, we compared the dosimetric and delivery parameters for plans generated with both types of delivery systems to determine if there were any significant differences between plans generated with the two MLC designs. In the second part of the study, we focused on the cases with significant difference, and studied how these MLC parameters contributed to those differences.Nine head and neck cancer patients previously treated at our institution were included in this study under an Institutional Review Board (IRB)-approved retrospective study protocol.Overall plan quality evaluationAll nine cases were planned in parallel using combinations of the Truebeam with Millennium 120 (TB MLC) and the Halcyon system with the dual-layer MLC system as well as two planning techniques: IMRT and VMAT. For the beam arrangements, all cases used nine equally spaced beams for IMRT and two full or half arcs for VMAT. Half arcs were only used for two unilateral post-operative cases to reduce dose spread to the contralateral neck, which is our standard institutional approach in such patients. The same isocenter was used across all different planning techniques and platforms. Plans were generated by experienced physicists and dosimetrists and subsequently reviewed by a single attending physician to ensure the quality of the plans meet clinical requirements. To simulate how a clinical plan would be generated, 6X was used for Truebeam and 6FFF for Halcyon, which is our standard beam energy choice in our clinical planning procedure. The planners were instructed to approach each plan as a separate clinical case and to achieve overall the best plan without comparing between platforms. All plans were performed with the Eclipse™ treatment planning systems using Photon Optimizer™ and a pre-clinical Anisotropic Analytical Algorithm (AAA) v15.1 dose calculation algorithm with the same dose calculation grid size (2.5 mm). All plans for the same patients were normalized so that the same dose coverage (100% Rx dose covering 95% of PTV volume) was achieved for the highest prescribed target.Key dosimetric parameters of clinical interest were compared among different planning techniques and delivery systems: global maximal dose, maximum cord dose, and mean dose to the parotids, cochleae, esophagus, and larynx (non-target). Total monitor units required to delivery treatment were also compared. The paired treatment plans, i.e. plans created with different delivery system for the same patient, were compared statistically with the Wilcoxon signed-rank test. This comparison was done separately for IMRT and VMAT. Statistical significances  from this test were reported.Plan delivery time comparisonOne of the advantages of the Halcyon platform is that the gantry rotation speed is four times that of the Truebeam linac. This potentially enables plans to be delivered in a much shorter time and therefore increases efficiency and patient throughput. In this study the treatment delivery efficiency was evaluated by comparing the actual total beam-on time between the Truebeam and Halcyon platforms and between IMRT and VMAT plans. Five patients’ plans were included in this part of the study, each with four plans delivered: Truebeam-IMRT, Truebeam-VMAT, Halcyon-IMRT, Halcyon-VMAT. All plans were delivered with automation enabled, i.e. the machine automatically delivered all treatment fields without operator intervention, to mimic actual clinical usage.Impact of MLC parameters on plan qualityAn additional study was completed to identify the MLC parameters that contributed most to the observed differences in the plan quality. Because the Halcyon platform does not allow end users to modify the beam data within the treatment planning system, the TB MLC’s transmission, leaf speed, and DLG parameters were altered in the TB beam model configuration to  match with those of the Halcyon dual-layer MLC, as shown in Table . It shouPlan comparisonsIt is meaningful to compare global maximal dose (Dmax) under the same target coverage since all plans were normalized to have the same target coverage by the Rx dose. The left pane in Figure Another comparison shown in Figure OAR doses were compared for key dose-volume histogram (DVH) parameters and is visualized in Figure The paired differences between TB MLC and the dual-layer MLC plans are highlighted in Figure Wilcoxon signed-rank tests were performed between TB and dual-layer MLC plan pairs to determine the statistical significance of the observed differences, and the resulting p-values were included in Figure Plan delivery time comparisonPlan delivery time was evaluated and shown in Figure Another observation that can be made from Figure MLC parameters impact on OAR dosesIn this section, four types of hypothetical TB MLCs were simulated with DLG, leaf speed, and transmission, and all three of the above matched to the specifications of the Halcyon dual-layer MLC. Treatment plans were then re-optimized using these simulated MLC systems that resemble one or three parameters of the dual-layer MLC features.OAR dose reduction from simulated MLCs and the actual dual-layer MLC was extracted from re-optimized plans and visualized in Figure In this study we first compared Halcyon’s dual layer MLC’s performance in IMRT and VMAT treatment for head and neck cancer to that of Truebeam’s Millennium 120 MLC in terms of plan quality and delivery speed, and later identified which MLC parameter contributed the most to observed plan quality differences. Our finding on VMAT plan quality and delivery time comparison between Halcyon and Truebeam agrees with Michiels et al., who evaluated head and neck patients and concluded that Halcyon maintains plan quality for VMAT technique . The maiWhen comparing dual-layer MLC with TB MLC on OAR doses for IMRT plans, one could question whether Figure It was observed that the OAR dose reduction is more pronounced for IMRT plans than for VMAT plans. This is mainly due to higher MU used in IMRT to deliver same target dose compared to VMAT. Since target dose is the same, for a given MLC transmission factor higher MU results in more leakage radiation through the MLC reaching the OARs. Also because leakage radiation through the MLC is proportional to both the transmission factor and total MU, for a given transmission factor reduction, e.g. from TB to Halcyon MLC, more MU would result in more pronounced difference in the transmitted radiation beyond MLC, which directly contributes to OAR dose. This explains why the difference in OAR dose due to MLC transmission change is more pronounced for IMRT than for VMAT.It is evident from Figure 2 = 0.000049, or ~0.005%. This further reduces leakage and transmission dose to blocked areas, making the actual dual-layer MLC perform better than a single layer TB MLC with 0.007 transmission factor. There are other factors that were not simulated, such as the full range modulation and interdigitation, which are limited in TB MLC model. The ability of unrestricted leaf movement allows for additional capability to modulate beam intensity. This could explain why in some cases, the dual-layer MLC has lower mean normal tissue dose compared to simulated TB MLC, even with wider leaves. For Truebeam linacs with jaw-tracking capabilities, the dynamic jaws conforming to MLC openings may help reduce the MLC leakage to blocked regions albeit to a limited level since the jaws can only conform to the least extended  leaf position for any given aperture.On the other hand, because Halcyon MLCs are dual-layer stacked, the area under two layers of MLCs effectively experiences even less leaf leakage. The leakage with two layers blocking the beam can be approximated to be 0.007It should be noted that Halcyon MLC transmission factor used in this study was based on pre-clinical system that we had access to at the time of data acquisition. In the clinical release of Eclipse™ treatment planning system the transmission factor was changed to 0.047%. This will likely enhance the normal tissue sparing effect that was observed due to transmission factor being smaller than TB MLC.When compared to Truebeam's Millennium 120 MLC, the Halcyon treatment delivery system's newly designed dual-layer MLC is capable of generating head and neck IMRT/VMAT plans of similar quality despite having thicker MLC leaves. For some OARs, the IMRT plan using the dual-layer MLC provided better sparing in terms of mean dose. Detailed analysis showed that this improvement was largely due to a combination of lowered leaf transmission, faster leaf speed, and smaller DLG. Among these three MLC parameters, lower leaf transmission had the largest contribution for the improved OAR sparing. Thicker MLC leaves provided stronger mechanical construction, which resulted faster MLC movement speed. The delivery speed of head and neck treatment plans using Halcyon platform were nearly twice as fast than the TB MLC for both fixed gantry beam arrangement (IMRT mode) and VMAT mode.


Optic disc drusen (ODD) is an important clinical entity that is sometimes misdiagnosed as papilledema because of elevated and blurred disc margins. A 17-year-old male who presented with headaches underwent detailed ophthalmological examination as well as colored fundus photography, B-scan ultrasonography (USG), fundus autofluorescence (FAF), optical coherence tomography (OCT), optical coherence tomography angiography (OCTA), and visual field testing. His visual acuity was 10/10 in both eyes. Fundus examination revealed bilateral blurred and elevated optic disc margins. Diagnosis of bilateral ODD was confirmed with B-scan USG. FAF imaging revealed hyperautofluorescent areas on both optic discs. Optic nerve head OCT scans showed elevated irregular disc borders and thinning of the retinal nerve fiber layer in both eyes. On visual field testing, loss of the nasal visual field was detected in the left eye. OCTA imaging showed focal capillary dropout, especially in the nasal peripapillary area, in both eyes and reduced peripapillary and macular vessel density. In this case report, we evaluated the clinical findings and the structural features of bilateral ODD with multimodal imaging modalities including OCTA. Optic disc drusen (ODD) is the leading cause of this condition.5 A wide variety of imaging methods are used for diagnosis, including ultrasonography (USG), fundus autofluorescence (FAF), optical coherence tomography (OCT), fluorescein angiography (FA), and computed tomography (CT).7 With the recent introduction of optical coherence tomography angiography (OCTA), peripapillary and macular retinal vessels can now be visualized noninvasively without the use of contrast agents. This provides more detailed information about optic nerve head perfusion.8Superficial ODD are easily recognizable by the presence of yellow hyaline-like deposits on ophthalmoscopic examination. However, because deep or buried ODD  are not visible during examination, additional imaging methods are needed to differentiate from more serious conditions such as increased intracranial pressure and tumors.In this case report, we aimed to present the clinical features of a patient with ODD and the diagnostic methods used.A 17-year-old male presented with a 2-month history of headaches. The patient had no known systemic disease or history of trauma, drug use, or smoking. His family history was unremarkable, with no consanguinity. On ophthalmologic examination, his uncorrected visual acuity was 10/10 (0.0 LogMAR) in both eyes. His pupils were isochoric and light reflexes were normal in both eyes; no afferent pupillary defect was observed. Color vision test using Ishihara cards was normal in both eyes. Slit-lamp anterior segment examination was also normal. Intraocular pressure was 14 mmHg in the right eye and 16 mmHg in the left eye. Fundus examination revealed bilateral optic disc swelling and blurred disc margins .Visual field was evaluated using the Swedish Interactive Thresholding Algorithm (SITA) standard 24-2 threshold test on a Humphrey Field Analyzer III 750 (Zeiss Humphrey Systems) automated perimetry device. Scotoma was not detected in the right eye, while a significant visual field defect was evident in the inferonasal quadrant in the left eye . B-mode 9 ODD are typically deeply situated in very young children and may eventually become superficial in late childhood, around 12 years of age.10The prevalence of ODD is reported to be 0.2-2% in adults and 0.37-1% in children. The lower than expected prevalence in children has been attributed to difficulty in the use of imaging techniques in the diagnosis of deeply buried non-calcified drusen.11 It has been reported in the literature that vascular complications such as nonarteritic anterior ischemic optic neuropathy, choroidal neovascularization, and central retinal artery and vein occlusions may occur due to ODD, albeit rarely.12Although the pathogenesis is not known, drusen are believed to directly damage the retinal nerve fibers by axonal compression and indirectly cause ischemia in the nerve fiber layer as a result of vascular compression.13 reported that 76% of children presenting with suspected papilledema had pseudopapilledema. Misdiagnosis leads to unnecessary radiological imaging and invasive and expensive tests such as lumbar puncture or magnetic resonance imaging. Therefore, it is extremely important to be able to differentiate pseudopapilledema from papilledema, which has very different treatment, follow-up, and diagnosis. Fundus examination findings in favor of ODD are an absence of dilated capillary vessels over the disc, no blurring of the vessels around the disc, a nonhyperemic disc, and the absence of peripapillary RNFL thickening.4 Additional imaging methods are often needed to confirm the diagnosis.Kovarik et al.9 In accordance with literature, OCT revealed peripapillary RNFL thinning in our patient.B-mode USG remains the most reliable method for diagnosing ODD. While FAF imaging is useful for the diagnosis of superficial drusen, it can detect only 12-27% of buried drusen. OCT provides objective data through quantitative evaluation of the RNFL. Peripapillary RNFL thickness was reported to be greater in patients with papilledema compared to ODD. While RNFL values are often normal in patients with buried ODD, peripapillary thinning is observed in all quadrants in cases of superficial ODD.14 Visual field defects are less common in children (11-51%) than adults (50-90%). In patients with ODD, visual field defects have been related to older age, vision loss, and superficial, calcified drusen.9 In children, the most common visual field problems associated with ODD are nasal defects (54%), concentric narrowing (21%), and blind spot enlargement (18%). In addition, defects were reported to be more common in the inferonasal retinal nerve fiber bundles than superotemporal.15 Gaier et al.16 detected inferotemporal microvascular attenuation in OCTA consistent with a visual field defect in the superonasal quadrant in a patient with ODD. The authors also reported macular microvascular attenuation only in the superficial capillary plexus. Although our patient had a visual field defect only in his left eye, reduced vascular density in both eyes was detected, especially in the nasal peripapillary area. Macular vascular density was found to be reduced in the deep capillary plexus layer as well as the superficial capillary plexus. It was interesting that the areas of nonperfusion were different in the superficial and deep capillary layers. This may be related to the superficial location of the ODD in the left eye, which caused less compression of the deep capillary layer.Visual field defects have been detected in 73% of superficial drusen cases, compared to only 36% for buried drusen.16 Cennamo et al.17 reported that ODD patients had lower flow index and reduced vascular density on optic nerve head OCTA compared to the control group. In addition, OCTA findings were positively correlated with ganglion cell layer thickness on OCT, and the authors emphasized that flow rate measurements made with OCTA may be an early predictor of axonal damage in ODD patients.17 Unlike in other studies, our patient showed decreased vascular density on macular OCTA as well as optic nerve head OCTA. Therefore, areas of reduced vascular density detected by OCTA may be a predictor of future central scotoma. These findings support the hypothesis that enlarged ODD may cause acute or chronic ischemia by compressing nerve fibers or surrounding vessels. The prominent hyperautofluorescence on FAF, visual field defect, and RNFL thinning in our patient were compatible with superficial drusen. Additionally, the superficial capillary plexus was more affected than the deep capillary plexus on macular OCTA due to the superficial location. Macular OCTA findings also supported the FAF, visual field, and OCT outcomes.In recent studies, OCTA imaging of the optic nerve head has revealed capillary narrowing in the superficial capillary plexus layer, areas of capillary dropout, and decreased vascular density in ODD patients.In conclusion, OCTA has become more widely used for the evaluation of optic nerve pathologies as well as retinal diseases because it is a noninvasive, easy, fast, and practical method. OCTA can be used as an auxiliary diagnostic method to USG and FAF in the diagnosis of ODD. Although ODD is generally asymptomatic, OCTA evaluation of the optic nerve head and macula may play an important role in the early detection of ischemic complications. This should be further investigated in prospective studies with long-term OCTA follow-up of ODD patients.


How the brain decomposes and integrates information in multimodal speech perception is linked to oscillatory dynamics. However, how speech takes advantage of redundancy between different sensory modalities, and how this translates into specific oscillatory patterns remains unclear. We address the role of lower beta activity (~20 Hz), generally associated with motor functions, as an amodal central coordinator that receives bottom-up delta-theta copies from specific sensory areas and generate top-down temporal predictions for auditory entrainment. Dissociating temporal prediction from entrainment may explain how and why visual input benefits speech processing rather than adding cognitive load in multimodal speech perception. On the one hand, body movements convey prosodic and syllabic features at delta and theta rates . On the other hand, the natural precedence of visual input before auditory onsets may prepare the brain to anticipate and facilitate the integration of auditory delta-theta copies of the prosodic-syllabic structure. Here, we identify three fundamental criteria based on recent evidence and hypotheses, which support the notion that lower motor beta frequency may play a central and generic role in temporal prediction during speech perception. First, beta activity must respond to rhythmic stimulation across modalities. Second, beta power must respond to biological motion and speech-related movements conveying temporal information in multimodal speech processing. Third, temporal prediction may recruit a communication loop between motor and primary auditory cortices (PACs) via delta-to-beta cross-frequency coupling. We discuss evidence related to each criterion and extend these concepts to a beta-motivated framework of multimodal speech processing. Continuous speech perception shapes spontaneous oscillatory activity in neuron ensembles, ensuring coordinated signal processing between specialized areas for successful comprehension. It has been now established that the sampling of sensory input temporal structure relies on the brain’s low frequency entrainment (1–8 Hz) to relevant rhythmic features occurring at slow time scales, and present in both auditory and visual modalities  impose peaks of energy fluctuations at delta (1–3 Hz) and theta (4–8 Hz) rates in the speech envelope. These significant fluctuations of amplitude serve as relevant anchors for sensory areas to synchronize with incoming signal and drive low-level sensory processing. In the primary auditory cortex A1/A2, previous magnetoencephalography/electroencephalography (MEG/EEG) studies show that this entrainment is typically reflected by the synchronization of an optimal delta-theta phase and power of responding neuron ensembles with the onsets of prosodic and syllabic events occurring in speech. For instance, Gross et al.  investigHowever, speech is often multimodal with the onsets of the articulatory movements preceding the correspondent auditory onsets with a stable temporal delay Pilling, . This teper se using a target detection task where they modulated both temporal regularity and spectral dimensions in auditory tone sequences. They found that predictability of spectral context  increased the benefits of temporal predictability on target detection . In contrast, the effect of temporal regularity canceled out when a spectral prediction mismatch occurred, suggesting a hierarchy of predictions built on the perceptual context and increasing entrainment in sensory cortices. Further, Morillon and Baillet  beta activity must respond to biological motion in multimodal speech processing. As biological motion conveys equivalent information on the prosodic-syllabic temporal structure in multimodal speech, it is critical to establish that beta activity encodes temporal information conveyed in visual modality via speaker’s movements such in multimodal speech. Third, if beta-based temporal prediction modulates entrainment in the PAC, this should rely on a coupling between beta power bursts and the delta frequency. In the first place, auditory and visual prosodic structure representations generated in their respective sensory areas are integrated via the central amodal coordinator. Then, such delta-to-beta coupling would reflect descending beta-based feedback from motor cortex to auditory cortex to improve the delta phase alignment with prosodic cues in the incoming signal. In the current review, we report evidence regarding these three Recent research has addressed the role of lower beta power in unimodal stimulus-driven temporal prediction without direct motor engagement. Fujioka et al.  investigHowever, does lower beta power specifically support auditory temporal prediction, or support amodal mechanisms? In the latter case, lower beta power may also respond to visual stimuli. Recently, Keitel et al.  investigIn summary, lower beta activity entrains to temporally structured unimodal stimulation, fulfilling the first criterion for being an amodal coordinator supporting generic temporal prediction.Numerous studies reported beta activity in response to perception and execution of actions performed by the hands or the arms .How beta-based temporal predictions improve sensory processing should be reflected by correlated activities between low frequencies entrained to prosodic structure in the signal  and lower beta oscillations. The particular role of delta activity in  temporal predictions has been evidenced in recent studies  beta activity responds to temporal prediction in auditory, visual, and audiovisual (speech) stimulation. We also discussed the fact that (2) motor beta activity responds to biological motion, including non-verbal information in multimodal speech. Further there seems to be a functional overlap between biological motion perception and (sensory) temporal prediction in the lower beta band (~20 Hz). Lastly, (3) delta-to-beta cross-frequency coupling supports distant communication between (left) motor and auditory cortices when listening to speech. Meeting these three criteria, we hypothesize that lower beta plays the role of an amodal central organizer that coordinates afferent copies from visual and auditory areas in multimodal temporal prediction during speech perception. If so, a speaker’s speech-related movements  may provide a redundant copy of the temporal structure of the speech envelope at different timescales .Non-verbal information indeed translates the slow temporal structure of speech with a stable precedence  as a central coordinator of amodal temporal predictions during multimodal speech perception. We first reported evidence on beta engagement in unimodal temporal prediction and audiovisual integration. Second, we underlined the responsiveness of lower beta activity to biological motion and non-verbal information in audiovisual speech. Third, we described delta-to-beta cross-frequency coupling between left motor areas and auditory cortices, reflecting descending modulations on auditory entrainment. Based on these three fundamental criteria , we hypothesized that lower motor beta activity plays a central role in generic temporal predictions by driving the cooperation between converging sensory inputs via specialized areas and conveying feedback toward the auditory cortex to facilitate audiovisual speech perception.Ensembles of neurons that fire together at a frequency of 13–30 Hz. Beta activity can be recorded using EEG or MEG and is characterized by its phase in the cycle and its amplitude (reflected by the power). In the present review, lower beta activity corresponds to the 18–22 Hz frequency range centered around 20 Hz. While it is well established that beta activity in action perception and action originates in motor cortex , it is still unclear whether it also drives temporal predictions in sensation. As discussed here, the lower band of motor beta oscillations may represent a functional interface for entrainment and temporal prediction mechanisms in multimodal speech.When sensory areas respond to rhythmic stimulation, neural populations fire at the same frequency but not necessarily together in spontaneous activity. After a number of periodic repetitions, the neurons entrain their activity to the onsets of temporally predictable events to extract the temporal structure of a sensory input and improve the sampling of the incoming signal. Consequently, the neurons align their firing phase in the frequency cycle to the same onsets, which increases the coherence of overall activity when a future event occurs, Further synchronizing activity at the neural population level decreases noise , which is reflected in an increase of power in perception. Here we propose that in multimodal speech, entrainment to both body movements and verbal utterances provides two copies of the prosodic-syllabic structures (conveyed respectively at 1–3 Hz delta and 4–7 Hz theta rates) that converge toward a generic central coordinator for temporal prediction.We refer to feedback as descending information conveyed by lower beta activity from left motor cortex and improving entrainment in primary auditory cortex. Beta-based feedback on entrainment is likely supported by delta-to-beta cross-frequency coupling in motor and auditory cortices, reflected in beta bursts occurring at a delta rate.Continuous speech can be decomposed into different perceptual units occurring in distinct frequency ranges. The first one corresponds to speech envelope modulations and correlates with the syllabic rate that conveys information at 4–7 Hz (theta). The second dominant speech rhythm reflects regular modulations in the utterance contour of a speaker’s prosody. The prosodic modulations of supra-segmental features of speech  convey information at 1–3 Hz delta rate. Finally, additional acoustic information occurs at higher 30–50 Hz frequency range (gamma band) and reflects more subtle phonemic features such as fundamental formant variations.We use the term non-verbal information to define all types of body movements produced by a speaker and that potentially translate auditory features at different time scales. Lip movements, mouth aperture, and jaw constraints reflect sound production and occur at a syllabic rate (~4 Hz). In contrast, simple nods of other body parts  translate a speaker’s voice modulations and convey information at a prosodic rate (1–3 Hz). A speaker’s gestures also accompany speech prosody with rapid flicks that align with a speaker’s prosody.EB and SK prepared the manuscript draft. All authors approved the final manuscript.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.


A 74-year-old male patient was presented with scrotal swelling and a pulsatile mass of the left femoro-inguinal region. His medical history included hypertension, coronary artery disease, respiratory failure, and an aortobifemoral bypass surgery performed 7 years ago. Ultrasound evaluation revealed a massive scrotal hematoma. Computed tomography angiography (CTA) was conducted, confirming the aortobifemoral graft existence and revealing bilateral anastomotic pseudoaneurysms with the left one being ruptured, resulting in extension of the hematoma to the left femoro-inguinal region and the scrotum. An emergency surgery was performed, where proximal control of the left limb of the synthetic graft as well as distal control of the iliac vessels were accomplished. After the control of the hemorrhage, an iliofemoral bypass with a Polytetrafluoroethylene (PTFE) 6 mm synthetic graft was placed. Unfortunately, the patient passed away during the first postoperative day due to myocardial infarction. Taking into account the large number of reconstructive vascular procedures being performed in latest clinical practice, there is an expected increase in the number of post-procedural complications, including anastomotic pseudoaneurysm formation. If those anastomotic pseudoaneurysms are left untreated, they may lead to peripheral embolism or rupture, conditions that are limb or life-threatening respectively and in great need for immediate intervention . In thisA 74-year-old male patient came to our hospital's ER with a progressively enlarging scrotal edema of non-traumatic nature, noticed during the last few hours. Clinical examination at the Urology Department revealed hemodynamic stability and a painful pulsatile mass of the left femoro- inguinal region without any existing signs of local or systemic infection, causing however extreme discomfort to the patient. His past medical history included hypertension, coronary artery disease and respiratory failure, whereas his surgical history of interest included an aortobifemoral bypass surgery using a synthetic graft due to severe peripheral arterial disease, conducted 7 years ago. Ultrasound evaluation of the scrotum revealed scrotal hematoma existence. A triple phase computed tomography angiography (CTA) examination of the abdomen and pelvis was then performed using our Department's 64-detector row CT scanner  on an emergency basis, including unenhanced, arterial and portal phase post-IV contrast media administration. CT Angiography (CTA) protocol included supine patient positioning with hands above head and thin CT slices of 1.5 mm from the supraceliac aorta to proximal thighs. A total of 150 mL of non-ionic iodinated contrast material with iodine concentration of 370 mg/mL was used with a flow rate of 4 mL/sec, followed by saline flush. Both arterial and portal phase imaging were obtained using the fixed time-delay technique. Specifically, arterial phase CT angiographic images were obtained 25 sec after the onset of peripheral intravenous injection, whereas portal phase CT angiographic images were obtained 70 sec post contrast media injection. Maximum intensity projections (MIPs), oblique or curved multiplanar reformats and 3D volume-rendered (VR) images were also evaluated in addition to cross-sectional axial images. CTA showed the aortobifemoral graft and confirmed the clinical suspicion of a ruptured pseudoaneurysm at the distal end of the left limb of the graft , pseudoaneurysms represent by definition an arterial wall defect that allows luminal arterial blood communication with the adjacent soft tissue –4. They Although theoretically anastomotic pseudoaneurysms can develop at any anastomotic site, 80% of them appear at the femoro-inguinal region possibly because of acting forces from motion of the adjacent joint , 9. AnasIt is quite common for femoral pseudoaneurysms to measure up to 4 cm in diameter before being clinically identified and sometimes, they may exceed 15 cm before rupture . IndicatAccording to the current guidelines, CTA represents the reference non invasive imaging modality for the diagnosis of anastomotic pseudoaneurysms and their associated complications. It can also reveal signs of impending pseudoaneurysm rupture (fissure existence or lack of thrombus homogeneity), and help at therapeutic approach planning , 15. In Anastomotic pseudoaneurysm rupture constitutes a vascular emergency condition in great need for immediate surgical treatment, since there is always the risk of death due to excessive hemorrhage. Whenever a patient with a pulsatile mass and a medical history of vascular bypass surgery comes to the ER, clinical suspicion of the above complication should exist and a CTA must always take place on an emergency basis, as it highlights the pathology and it also helps decide the appropriate therapeutic approach.


Animal models of metastatic colon cancer are available but with varying degrees of reproducibility and several important limitations. We explored whether there were regional differences in the location of murine colon cancers and assessed the utility of murine models to explore the biological basis for such differences.Strong evidence reveals important differences between cancers in the proximal nu or NOD.Cg-PrkdcscidIl2rgTim1Wji/SzJ mice and compared the development of primary tumors and metastases.We re-analyzed data from our previous studies to assess the regional distribution of murine colon cancer. In survival surgery experiments, we injected HT-29 human colon cancer cells into the wall of the cecum or distal colon of Nu(NCr)-Foxn1Within 7–17 weeks after intramural cecal injection of HT-29 cells, eight mice failed to develop solid primary tumors or metastases. In contrast, within four weeks after cell injection into the distal colon, 13 mice developed metastases - 12 mice developed subcutaneous metastases; of these, four developed liver metastases and one developed both liver and lung metastases. One mouse developed liver metastases only. Histological examination confirmed these lesions were adenocarcinomas.Our findings reveal the preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon. The new approach of injecting cells into the distal colon wall results in a pattern of colon cancer development that closely mimics the progression of metastatic colon cancer in humans. This novel model of colon neoplasia has great potential for exploring anatomical differences in colon cancer and testing novel therapeutics. Metastatic cancer is the chief reason for CRC-related death; primary tumors without metastases are readily cured by endoscopic or surgical therapy. Intriguingly, strong evidence reveals important differences between cancers in the proximal vs. distal colon–6.In developed countries, colorectal cancer (CRC) is the second most common cause of cancer-related death in men and the third in womenAPC, K-ras, DCC, and p53. Previous studies reported conflicting findings with regards to whether mortality was significantly different in those with primary right- vs. left-sided colon cancer–9. A meta-analysis found higher mortality in patients with right-sided compared to left-sided colon cancer. A recent database study found that right-sided colon cancer was associated with lower cancer-specific mortality at the localization stage, equivalent mortality at the regional stage, and higher mortality at the metastatic stage. Another recent retrospective study found those with left-sided colon cancer had better survival outcomes, especially with stage III cancers. From 1998 to 2013, the SEER  database identified 90,635 and 112,679 persons diagnosed with left- and right-sided colon cancer, respectively.Compared to cancers of the distal colon, proximal colon cancers are more common in women, are associated with microsatellite instability and the serrated pathway, and are more likely to be at advanced stages when first diagnosed. Distal colon tumors are more likely to be associated with chromosomal instability and arise from the pathway involving dysregulated Few therapeutics are either effective or available to treat persons with metastases to the liver and other organs. To improve therapeutic outcomes, there is great urgency to gain a better understanding of the mechanisms underlying colon cancer dissemination as a basis to develop targeted therapies. For investigators to test such new therapeutics with some degree of reliably there is also a great need to conceive and develop novel models that more closely mimic human disease.; in the most commonly used ApcMin mouse strains, tumors are almost uniformly adenomas, not adenocarcinomas, and are located predominantly in the small intestine, not colon. Also, murine models using injection of human colon cancer cells are limited by the need to use immune-deficient mice to allow tumors to develop, thus excluding the testing of immunotherapies. Nonetheless, using syngeneic models with murine colon cancer cells is also imperfect because these cell lines are less well-studied and their biology may not mimic that of human cancers.Several animal models of metastatic colon cancer are available, with varying degrees of reproducibility, limitations, and imperfect fidelity to the biology of human cancer. Current murine models are limited by location, depending on what model is used, and cancers in different locations have different genetic profiles. A case in point is ApcMin mouse models that were meant to recapitulate defective Wnt/β-catenin signaling present in ~90% of human colon cancer. These include models employing chemical carcinogenesis, genetic engineering, and animal- or patient-derived xenografts,13; the latter have been particularly helpful to study the mechanisms underlying the metastatic spread of human colon cancer and identify susceptible therapeutic targets,12. Colorectal cancer xenografts grown subcutaneously in immunodeficient mice are limited by the lack of metastasis; instead, orthotopic tumor models involving injection of CRC tumor cells or implantation of tumor tissue directly into the wall of the colon have the potential to be more representative of human metastasis.Despite their limitations, murine models have long served as the most reliable platform for preclinical evaluation of new drugs and technologiesvs. distal colon of immune-deficient mice.We sought to determine if mice could serve as a model to explore regional differences in the location of cancers within the colon. To seek such differences in the growth and progression of colon neoplasia in mouse models, we first determined the location of colon tumors in mice treated with a colon-selective carcinogen or with a genetic predisposition to intestinal neoplasia. Next, based on our initial findings, we compared the proclivity of human colon cancer cells to grow and invade the proximal –19. During this interval, we had treated 10- to 23-week-old male mice on a variety of genetic backgrounds with weekly intraperitoneal injections of 7.5 mg azoxymethane (AOM)/kg body weight for 4 weeks. In C57BL/6 mice that are resistant to AOM treatment alone, we supplemented the drinking water with 2.5% dextran sodium sulfate (DSS) for 5 days. We euthanized mice 20 weeks after the first AOM injection. An investigator masked to mouse genotype and treatments measured tumor number and size, and tumors were characterized as adenomas or adenocarcinomas based on size, contour, and color. A senior pathologist classified colon tumors as adenomas or adenocarcinoma based on consensus recommendations.To assess the regional distribution of murine colon cancer, we re-analyzed data from our published and unpublished murine colon cancer studies conducted from 2006 through 20182 and passaged weekly at subconfluence after trypsinization. We suspended cells in DPBS (50 × 106 cell/mL) containing 10 μmol/L Y27632 and 50% Matrigel.We purchased authenticated HT-29 cells from American type culture collection (ATCC). HT-29 cells were grown in McCoy’s 5A medium (Life Technologies) supplemented with 10% FBS. We grew cells in a humidified incubator at 37 °C with 5% COnu (nude) mice and NOD. Cg-PrkdcscidIl2rgTim1Wji/SzJ (NSG) mice, obtained from both the University of Maryland Veterinary Resources and Jackson Laboratories .All animal studies were conducted at the Baltimore VA Hospital Animal Facility and our laboratory in the Bressler Research Building at the University of Maryland School of Medicine. All surgical procedures were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee under the Office of Animal Welfare Assurance. The Research and Development Committee at the Baltimore VA also approved animal studies. We used 11- to 14-week old male Nu(NCr)-Foxn1In a biosafety cabinet (BSL2) we anesthetized mice with continuous vaporized isoflurane for general anesthesia and performed laparotomy and cell injections with the mice on a warming pad. After confirming a sufficient level of anesthesia by a toe pinch, we positioned mice prone. For corneal protection, we applied lubricant  to each eye. We dis-infected the mouse’s upper back with an alcohol swab and administered buprenorphine SR  or carprofen  subcutaneously for analgesia. We then placed mice supine and, if necessary, clipped the anterior abdominal hair. After skin preparation with alcohol, to provide local anesthesia we injected mice subcutaneously with 0.25% bupivacaine  along the planned midline laparotomy site. Next, we cleansed the abdomen with povidine-iodine solution and alcohol and applied sterile drapes. We made a small midline laparotomy and inserted a self-retaining retractor in the upper abdomen.Following injection of human colon cancer cells and replacing the intestines, we approximated fascial edges with 5–0 vicryl running sutures and closed the skin primarily with 4–0 nylon interrupted sutures. After applying skin glue  to the suture line, we awakened mice slowly from anesthesia and placed them in a clean cage for recovery with close monitoring. After completion of the operation, the mice were administered analgesia for at least 72 h post-operatively and monitored closely.6 (40–100 μL) HT-29 human colon cancer cells into the cecum of nude or NSG mice. We chose these cell numbers based on previous reports describing successful metastatic models of colon cancer in mice,12,22,23. We described pre-operative steps above. The cecum was located using moist sterile cotton tip applicators and brought outside the abdomen onto a moist 2 × 2 sterile gauze. In all mice, we injected 2–5 × 106 cells (40–100 μL) into the wall of the cecum using a 27-guage needle. After injection, we applied light pressure at the injection site for approximately 30 s with a moist sterile tip applicator and inspected the area for leakage. We irrigated the cecum and abdominal cavity with warm DPBS, and then returned the cecum to its normal anatomic position within the abdomen. Closure of the abdomen was performed as describe above.To explore the predilection of human colon cancer cells to form tumors in different regions of the mouse, we first injected 2 to 5 × 106 cells (40 μL) in each flank. We recovered mice from anesthesia in their cages.In mice failing to form cecal tumors, we confirmed the ability of the HT-29 cells to form xenografts and metastases following subcutaneous and splenic injection, respectively. For subcutaneous injections, we briefly anesthetized mice with vaporized isoflurane, disinfected their flanks with alcohol, and injected 2 × 106 cells (100 μL) into the wall of the spleen using a 27-guage needle. After injection, we applied light pressure at the injection site for approximately 30 s with a moist sterile tip applicator and inspected the area for leakage and bleeding. We irrigated the spleen and abdominal cavity with warm DPBS, and then returned the spleen to its normal anatomic position within the abdomen. After 1 h, we removed the spleen and irrigated the abdomen again with DPBS. We closed the abdomen as described above.We described pre-operative steps above. The spleen was located using moist sterile cotton tip applicators and brought forward within the abdomen. In all mice, we injected 5 × 106 (100 μL) HT-29 human colon cancer cells into the wall of the distal colon of nude or NSG mice. We described pre-operative steps above. The distal colon was located using moist sterile cotton tip applicators , all tumors were limited to the distal half of the colon; no proximal lesions were present  developed a cecal lesion . Yet, wi,23,24, we euthanized mice four weeks after cell injection. Twelve mice developed subcutaneous anterior abdominal metastases; of these, four developed liver metastases and one developed both liver and lung metastases [Within four weeks after cell injection, 12 mice developed primary colon tumor at the injection site in the distal colon and 13 mice  developed metastases . Based otastases . One moutastases , in the tastases , in the tastases , in the tastases , and multastases .,26. Whether the primary tumor is located in the right or left colon has been reported to play a prognostic role in metastatic colorectal cancer, although new findings suggest primary tumor ‘sidedness’ may be a less important determinant of overall and disease-specific survival than patient characteristics and other pathological features.Increasing evidence supports the presence of major differences in right- and left-sided colon cancers with regard to the host’s clinical characteristics, microbiome, response to treatment and outcome. Although molecular and genetic profiling of cancer cells has revealed some important differences, the reasons for this anatomical disparity remain unclearOur studies reveal that mice appear to be a suitable model to explore the predilection of cancer for different anatomical regions of the colon. We found that mice treated with a colon-selective carcinogen or with a genetic predisposition to intestinal neoplasia developed tumors only in the distal half of the colon; there were no proximal lesions. Likewise, immune-deficient mice preferentially developed colon cancer and metastases when we injected HT-29 human colon cancer cells into the distal, rather than proximal, colon.in vitro model of human colon cancer and express M3 type muscarinic receptors (M3R), a focus of our research program–29. Injecting HT-29 human colon cancer cells between the mucosa and the muscularis external layers of the distal colon wall of immunodeficient mice resulted in a pattern of tumor dissemination that mimicked human disease. Nonetheless, while it is uncommon for colon cancer in humans to spread to the skin and subcutaneous tissues, we found that when injected in mice, HT-29 cells are capable of diffuse metastasis, including to the skin. At present, we cannot explain why these colon cancer cells had a predilection for the anterior abdominal subcutaneous tissue. The location of these skin metastases near the surgical incision site leads us to speculate that features of the inflammatory response to the skin incision  may attract migrating colon cancer cells to this location, a testable hypothesis that may expand our understanding of the biology underlying tumor metastasis. We will test this hypothesis in future studies.In the course of these studies, we developed a novel method for inducing metastatic colon cancer in mice. We chose HT-29 cells for our studies because they a are commonly used in vitro studies and useful to study and test novel therapies that target M3R and its downstream signaling pathways to attenuate cancer cell dissemination. This model is relatively straightforward and the procedures easy to learn and perform by an investigator experienced in animal surgery, with reasonably rapid development of primary solid tumors and metastases. Unlike xenograft models, this method requires only one mouse and one operation to generate both colon cancer and metastasis. Our approach appears more biologically relevant than models in which investigators inject cells into the tail vein or footpad.We believe this novel animal model will be an important adjunct to our . Yet, even these humanized models are likely to present important obstacles with regard to mimicking the physiological maturation of human immune cells and the progression of human colon cancer.An important limitation of our new approach is that, as discussed in the Introduction, colon cancer immunotherapy cannot be studied in models using immune-deficient mice. However, humanizing the mouse immune system may achieve this goal. Next-generation models, including “immunoavatar mice” could offer the ability to study the effects of immunotherapy in colon cancer. Hemato-lymphoid humanized mouse models may allow the development of a complete human immune system in a human tumor-bearing mouseIn conclusion, our findings identify preferential growth of murine colon neoplasia and invasive human orthotopic xenografts in the distal mouse colon. These data support the utility of mouse models to study anatomical variance in the development and progression of colon neoplasia. We describe a useful model for inducing metastatic colon cancer in mice that is neither laborious nor time-consuming. This novel approach furnishes animals that closely mimic the progression of metastatic colon cancer in humans. This approach shows promise for studying novel therapeutics targeting colon cancer dissemination and metastasis.