Electrophysiological analysis confirmed the existence of synaptic inputs into the pACC by pIC GABAergic cells. Optogenetic stimulation of these pathways did not change mechanical nociception, but induced conditioned place preference behavior responses. Our results suggest the presence of inhibitory projections between the pIC and the pACC which are able to selectively modulate affective aspects of neuropathic pain.The insula and anterior cingulate cortex (ACC) are brain regions that undergo structural and functional reorganization in neuropathic pain states. Here, we aimed to study inhibitory parvalbumin positive (PV+) posterior insula (pIC) to posterior ACC (pACC) projections, and to evaluate the effects of direct optogenetic manipulation of such projections on mechanical nociception and spontaneous ongoing pain in mice with Spared Nerve Injury (SNI). CTBThe online version contains supplementary material available at 10.1186/s13041-023-01052-8. The pain modulatory system comprises anatomical pathways that include inhibitory circuits originating from cortical areas such as the insula and anterior cingulate cortex (ACC) . The pos488 was injected into the pACC region of PV-tdTomato mice, and overlap between CTB488 and tdTomato label in slices from the insular cortex visualized and quantified. The green retrotracer-labeled GABAergic cells in the pIC are shown in Fig. 488 injection was verified by examination of pACC slices . We also examined the degree of c-Fos activation in GABAergic cells projecting to the pACC in the presence and absence of SNI . Hence, SNI does not appear to induce increased activity in GABAergic pIC to pACC projections.We first examined to what extent the insular cortex and pACC are linked by inhibitory connections. CTBces Fig. b. Quantices Fig. c revealsTo complement our retro-tracing experiments, we used electrophysiology on pACC slice preps with opto-stimulation of synaptic inputs from the pIC. AAV-EF1a-DIO-ChR2-H134R was injected in the pIC of PVCre transgenic mice to express ChR2 in PV+ neurons . PVCre SHAM and SNI mice were subjected to optostimulation with a blue laser for selective ChR2 activation in PV+ projections from the pIC to the pACC. There was no effect of optostimulation on mechanical withdrawal thresholds in SHAM mice  (Fig. Several interconnected brain regions have been implicated in neuropathic pain, including the prefrontal cortex , 11, amyOnly a small portion of pIC to pACC projections were activated under basal conditions, and the proportion of activated cells appeared even lower after SNI surgery. c-Fos labeling has limitations, including a lack of quantitative output of neuronal activity, and subtle activity alterations within a specific subset of already active neurons due to SNI may not have been resolved. Our sample size was, however, limited, and thus putative activity differences between SHAM and SNI surgery may not have been fully resolved.In summary, our results support the idea that inhibitory pathways between the pIC and pACC can modulate affective-motivational aspects of pain, highlighting these areas as potential targets for the management of neuropathic pain.Additional file 1. Materials and Methods


De novo spinal dural arteriovenous fistulas (AVFs) have been reported as metachronous AVFs However, metachronous spinal AVFs are extremely rare, and their pathogenesis remains uncertain. We report a case of de novo radicular AVF (RAVF) following treatment for spinal AVF at the craniocervical junction (CCJ). We also reviewed the literature and discussed the pathogenesis of metachronous spinal AVF. A 64-year-old male patient diagnosed with spinal AVF at the CCJ supplied from the right C1 segmental artery was treated with Onyx-18  trans-arterial embolization, resulting in partial occlusion. Angiography showed a slight residual shunt two weeks after the embolization without another shunt lesion. A five-year follow-up spinal angiography showed de novo RAVF at the C4 level and complete occlusion of the first AVF. The second AVF was not treated because it was asymptomatic, and the patient remained asymptomatic. De novo RAVF was found to develop five years after the embolization of a CCJ-spinal AVF in a patient. This is the first case of de novo RAVF post-treatment of a spinal AVF. This case demonstrated that RAVF could develop as an acquired disease. De novo spinal dural arteriovenous fistulas (AVFs) have been rarely reported as metachronous AVFs without a genetic background . SecondaHistory and initial treatmentA 64-year-old male patient had visited another hospital complaining of right trigeminal neuralgia and gait disturbance for two weeks. Brain MRI showed an abnormal flow void at the anterior surface of the medulla oblongata and varix with perifocal edema at the right medulla oblongata Figure , 1B. CerTrans-arterial embolization with Onyx-18  was performed two months after onset, resulting in partial shunt occlusion. Transient ischemic symptoms occurred postoperatively because of thrombosis of the varix and venous infarction of the right middle cerebellar peduncle. The right VA angiography performed two weeks postoperatively showed a slight residual shunt Figure , 2B, andFollow-upMRI showed no sign of recurrence of the treated shunt lesion for the follow-up period of four years. However, the MRI performed five years after treatment showed abnormal vessels on the anterior surface of the cervical spinal cord. The subsequent spinal angiography showed complete occlusion of the treated CCJ-AVF five years later Figure . HoweverHerein, we report a de novo RAVF without a hereditary background. It was angiographically demonstrated five years after spinal AVF embolization, away from the first lesion. To the best of our knowledge, there are no previous reports of de novo RAVF proven angiographically, and this case provides new insights into the pathogenesis of the RAVF of an acquired nature.Reports of multiple spinal AVFs are rare, and most of them were diagnosed synchronously or on the second follow-up examination. Some of these second lesions were missed during the initial angiography, probably because a complete spinal angiography was not performed -15. De nPrevious trauma, spinal surgery, and inflammation have been reported as causes of the formation of a spinal AVF -9. SpeciRegarding diagnosing RAVFs, it is often difficult to precisely identify the location of the shunt in the spinal nerve root by a single diagnostic modality, including CT, MRI, and angiography. Therefore, some previously reported spinal dural AVFs or perimedullary AVFs may be RAVFs, especially at the cervical level. We identified the location of the shunt by creating a fusion image between high-resolution thin-slice MRI and maximum-intensity projection images of rotational spinal angiography, as we reported elsewhere .Delayed clinical deterioration following a short period of clinical improvement has been reported after the treatment of spinal dural AVFs . In suchThis is a single case report, but this first case of de novo RAVF developed after embolization of a spinal AVF indicates that spinal RAVF can be an acquired lesion like a spinal dural AVF. A patient should be followed-up carefully after treatment for spinal AVF, considering the possibility of the development of de novo spinal AVF, including RAVF.


Cognitive dysfunction is common in Parkinson’s disease (PD) and is diagnosed by complex, time-consuming psychometric tests which are affected by language and education, subject to learning effects, and not suitable for continuous monitoring of cognition.We developed and evaluated an EEG-based biomarker to index cognitive functions in PD from a few minutes of resting-state EEG.We hypothesized that synchronous changes in EEG across the power spectrum can measure cognition. We optimized a data-driven algorithm to efficiently capture these changes and index cognitive function in 100 PD and 49 control participants. We compared our EEG-based cognitive index with the Montreal cognitive assessment (MoCA) and cognitive tests across different domains from the National Institutes of Health (NIH) Toolbox using cross-validation schemes, regression models, and randomization tests.p value < 0.001 with MoCA; rho ≥ 0.56, p value < 0.001 with cognitive tests from the NIH Toolbox) outperforming traditional spectral markers (rho = −0.30 – 0.37). The index showed a strong fit in regression models (R2 = 0.46) with MoCA, yielded 80% accuracy in detecting cognitive impairment, and was effective in both PD and control participants.We observed cognition-related changes in EEG activities over multiple spectral rhythms. Utilizing only 8 best-performing EEG electrodes, our proposed index strongly correlated with cognition (rho = 0.68, Our approach is computationally efficient for real-time indexing of cognition across domains, implementable even in hardware with limited computing capabilities, making it potentially compatible with dynamic therapies such as closed-loop neurostimulation, and will inform next-generation neurophysiological biomarkers for monitoring cognition in PD and other neurological diseases. Because PD-related cognitive symptoms can predict morbidity and mortality, early diagnosis may help counsel families and guide crucial treatment decisions. Detailed neuropsychological testing5 is the gold standard for determining cognitive function. However, neuropsychological testing requires several hours and trained examiners and is subject to learning effects, especially with frequent testing6. Therefore, it is not readily compatible with repeated measurements throughout the day or even within short time intervals such as days or weeks. These features limit its role in capturing cognitive fluctuations in PD or real-time feedback for neuromodulation therapies where cognition can be negatively affected while improving motor function9.Cognitive dysfunction is a major non-motor symptom of Parkinson’s disease (PD), affecting ~ 20% of the individuals with PD at initial diagnosis and leading to dementia in more than 80% of the individuals with disease progression and aging10 or computerized batteries such as National Institutes of Health Toolbox Cognitive Battery11 (NIH-Toolbox) have similar limitations. MoCA is a high-performing screening tool for detecting PD-related cognitive impairment15. Computer-based cognitive tests provided by the NIH-Toolbox are also commonly used as standardized measures of cognition for insights into different cognitive domains16. While these cognitive measures can be readily measured, they depend on paper or digital text input formats limited by the patient’s motor and language proficiency, as well as by trained examiners during data collection and scoring17.Short screening tests such as Montreal Cognitive Assessment (MoCA)19. EEG-based indexing of PD-related cognitive functions can generate objective, rapid, and robust measures of cognition with continuous measurements in complex environments. More importantly, it can contribute to easily repeatable longitudinal assessments of cognition with disease progression, provide a screening process for cognitive profiling of the patients, and refine PD treatments such as closed-loop neurostimulation for avoiding cognitive side effects and improving cognitive symptoms20. In previous studies, EEG-based analysis has facilitated a more accurate characterization of granular fluctuations and it is sensitive to the abnormalities in cortical function that precedes the occurrence of overt disease manifestations22. EEG recording procedure is generally less stressful and well-tolerated by individuals with cognitive impairments. In addition, EEG-based cognitive measures are more objective and reproducible compared to neuropsychological testing due to the inherent variations in test procedures and inter-rater variabilities21. Thus, cognitive assessment by readily available EEG that does not require expert administration and is compatible with repeated or continuous measurement would be of great benefit. Changes in EEG activities correlate with cognitive measures such as MoCA and cognitive tests from NIH-Toolbox in various medical conditions23. However, to date, few EEG-based biomarkers have been found in PD patients that correlate strongly with cognitive performances measured by these tests even though such EEG-based measures can achieve superior assessment of cognitive fluctuations24.EEG provides a widely available, low-cost measure of cortical neurophysiology which can provide predictive electrophysiological markers for monitoring instantaneous and long-term cognitive functioning29, most prior EEG-based approaches have focused on specific spectral ranges leading to a limited capturing of EEG activities related to cognition. We recently developed a novel data-driven approach termed Linear predictive coding EEG Algorithm for Parkinson’s Disease (LEAPD) that can efficiently capture spectral EEG profiles over a broad frequency range and reliably detect PD and PD-related depression31. In this study, we hypothesized that cognition-related changes in EEG activities occur in multiple spectral rhythms across the power spectrum in PD and efficient capturing of these changes can index cognitive functions. We utilized LEAPD for capturing these EEG spectral changes related to cognition in a holistic fashion and developed an EEG-based biomarker to index cognition in PD. Our goal was to develop a cognitive assessment tool based on the neurophysiological changes in cortical activities as observed by EEG which would be quick, independent of any specific language and trained expertise while leading to a better understanding of the neurophysiology of cognitive impairment in PD.While changes in EEG over multiple spectral rhythms, power ratios, and phase-couplings can correlate with cognition and achieve moderate correlation with several psychometric testsIn this study, we collected resting-state EEG from 100 individuals with PD and 49 control participants, conducted traditional spectral analyses, and optimized LEAPD to estimate general cognitive function as measured by the MoCA and domain-specific cognitive function using tests from NIH-Toolbox. Our spectral analyses revealed synchronous changes related to cognitive measures over a broad spectrum in EEG. We found that LEAPD can efficiently capture these changes, strongly correlates with MoCA scores as well as with cognitive scores from NIH-Toolbox, and can detect MoCA-defined cognitive impairment in PD and control participants. These results not only provided evidence supporting our hypothesis on cognition-related changes in EEG activities but also delivered cognitive measures based on changes in cortical activities. Specifically, strong correlations with MoCA and other well-established cognitive measures indicate the effectiveness of EEG to quickly gauge cognitive impairment that can lead to EEG-based diagnostic technologies for detecting cognitive outcomes quickly and accurately, real-time feedback on the cognitive effects of medical and surgical interventions, and long-term monitoring of cognitive function in PD and other neurodegenerative diseases.https://icts.uiowa.edu/star). The motor component of the United Parkinson’s Disease Rating Scale and the MoCA scores were administered by trained raters. Participants also completed other clinical assessments of cognition, mood, and gait. The study was approved by the University of Iowa Institutional Review Board (protocol # 201707828). Written informed consent was provided by all participants. We used MoCA to quantify cognitive condition among participants as it is more sensitive to cognitive deterioration in PD 14. We defined cognitive impairment as MoCA scores < 26 and cognitively normal as MoCA scores 26–3033. Among all 149 participants, 53 PD and 11 control participants had MoCA-defined cognitive impairment (MoCA score < 26). The control participants with cognitive impairment had no pre-existing neurological or psychiatric disease. The median MoCA score among all participants was 26. To assess specific aspects of cognitive functioning, we used NIH-Toolbox, a standardized computer-based neuropsychological screening battery11. Five tests from the NIH-Toolbox were administered to the participants: Picture vocabulary test (PVT), Pattern comparison processing speed test (PCPST), Dimensional change card sorting test (DCCST), Flanker inhibitory control and attention test (FICAT), and Picture sequence memory test (PSMT). The tests were administered through iPads and extensive user directions were provided by the examiner.We recruited 100 PD participants from the Movement Disorders Clinic at the University of Iowa, Iowa City between 2017 and 2022 (Table 1). A movement disorders physician diagnosed each individual with PD according to the United Kingdom Brain Bank criteria. All procedures were performed while participants were taking their usual medications. Additionally, 49 demographically similar controls without known neurological disease were recruited from the general Iowa City community between 2017 and 2022 through the Seniors Together in Aging Research registry  and cognitively normal participants . To optimize training models for each of the cognitive tests from NIH-Toolbox, all participants were similarly divided into two respective training groups based on their scores (median as cut-off) while the classification of normal cognition and cognitive impairment was based on only MoCA scores.Resting-state EEG was collected from participants while they sat in a quiet room with their eyes open for a few minutes ON dopaminergic medications as usual  within 131. The key aspect of LEAPD is the use of linear predictive coding (LPC) for encoding EEG time series and efficiently capturing its unique spectral profile by capturing dominant oscillatory modes and encoding them into a few LPC parameters34. In this study, we utilized LEAPD to capture cognitive function by encoding EEG data using LPC and finding separate affine subspaces for cognitively impaired and cognitively normal participants. We hypothesized that after encoding EEG into vectors of LPC coefficients, there are two distinct affine subspaces  such that the relative distances from the two affine subspaces (LEAPD indices) highly correlate with cognition and can detect cognitive impairment.  were performed using MATLAB 2021b and R (version 4.1.2). All data and statistical analyses were reviewed by the Biostatistics and Research Design Core in the Institute for Clinical and Translational Science at the University of Iowa.As the distribution of the bounded LEAPD index is non-gaussian, we utilized the Spearman (rank) partial correlation method controlling for the participants’ age to measure correlation while accounting for group-level age differences (Table 1) 33. Spectral analyses of EEG revealed moderate correlations with MoCA scores , increased alpha (8 – 13 Hz) power in parietal region , increased gamma (31 – 100 Hz) power in central-parietal region , increased delta (1 – 4 Hz) power in central-parietal region  and reduced theta (4 – 8 Hz) power in left frontal region . For investigating the correlation between spectral ratios and MoCA scores, we chose the alpha/theta log-spectral ratio29 which simultaneously captures reduced theta and increased alpha power and showed a statistically significant correlation in almost all regions with MoCA  across all cross-validation schemes . MoCA score correlations with LEAPD indices were two times higher and significantly stronger than those from spectral analyses . MOCA and LEAPD index correlations were also twice as high as the highest previously reported MoCA correlation achieved by EEG features in PD24. Correlations with MoCA scores were similar (rho = 0.66 ± 0.024) for participants with PD alone, but lower in control participants . During the randomization test where we shuffled MoCA scores among all participants, MoCA correlations obtained by LEAPD were very low and not statistically significant .Next, we tested the hypothesis that LEAPD, which captures the spectral profile of EEG over a broad range, can measure cognitive function in PD. Individual electrode performances demonstrated that LEAPD indices from single electrodes strongly correlated with MoCA score . Differep value <0.005). PCPST, PSMT and DCCST scores correlated with decreased theta and increased beta power with the highest correlation of −0.29 , −0.30 , and 0.23  respectively. Alpha, beta and gamma power showed significant correlations with FICAT scores with the highest correlation of 0.36 . In contrast, the combined LEAPD index showed high correlations with the cognitive scores across all cross-validations . No statistically significant correlation was found between LEAPD and NIH-Toolbox cognitive scores in the randomization test for all participants (Supplementary Table S2). While we presented correlation results using Spearman’s rho, we observed similar results with Pearson correlation. Taken together, these data demonstrate that LEAPD index could strongly correlate with cognition in PD.We also correlated LEAPD and EEG spectral powers with cognitive scores from the NIH-Toolbox . Alpha and beta power correlated with PVT scores in the parietal region with the highest correlation of 0.24  while the top performing traditional spectral features yielded moderate AUC , but lower AUC and sensitivity . In the randomization test with MOCA-shuffled data, the results were close to chance . Finally, we checked the potential effects of the group sizes by subsampling the dataset with equal numbers of cognitively impaired and cognitively normal participants in leave-one-out cross-validation which provided similar performances  to the full dataset (Table 2).For all participants, LEAPD classified cognitive impairment and normal cognition with 0.90 ± 0.01 AUC (area under the receiver operating curve), 80.44 ± 0.55% classifier accuracy, 79.52 ± 0.65% sensitivity, and 81.14 ± 0.99% specificity across all cross-validation schemes  across all cross-validations, and the quadratic model had a statistically significant better fit compared to the linear model . Regression models for MoCA-shuffled data were not statistically significant (Supplementary Table S2).We noted that the correlation between the combined LEAPD indices and MoCA scores was unlikely to be linear. To explore this relationship, we used regression analyses. Both linear and quadratic regression models for the combined LEAPD index and MoCA scores were statistically significant (linear and quadratic 2 for PD participants compared to control participants (Table 2). No statistically significant effect of PD was observed after including it in the quadratic model as a random effect . No additional assumptions were made during the regression analyses. These data suggest a quadratic relationship between the combined LEAPD index and MoCA scores that is statistically independent of PD, and that the algorithm might be triggered by aspects of the EEG signals related to cognitive status, irrespective of PD.Quadratic regression models achieved higher RTo investigate the robustness of LEAPD, we examined how correlation and classification performances vary with the number of electrodes . The correlation of the combined LEAPD index with MoCA score and the AUC of cognitive classification both reached their respective maximums with 51 electrodes while the maximum classifier accuracy was achieved with 16 electrodes . However31, can efficiently capture EEG spectral profiles for cognition and detect cognitive dysfunction both in PD and control participants. Our data showed that by rapidly encoding resting-state EEG data from eight electrodes, LEAPD can accurately index cognitive function as measured by MoCA or by the cognitive tests from the NIH toolbox. It also detected cognitive impairment in control participants, performed consistently across multiple cross-validation schemes, provided reliable performance during data truncations, and showed no indication of overfitting in the randomization tests. The lack of significant correlation between LEAPD and cognitive tests during training and validation using data with randomly-shuffled cognitive scores showed the dependency of LEAPD’s performance on capturing the neurophysiological changes related to cognition. Thus, LEAPD can be useful for finding novel EEG-based biomarkers for cognitive impairment in humans including individuals with PD.In this study, we hypothesized that EEG activities change in a wide frequency range across the power spectrum with cognition and capturing these changes can lead to EEG-based cognitive measures. Our spectral analyses indicated that EEG activities over multiple spectral rhythms correlate with cognitive measures. We found that LEAPD, a data-driven machine learning algorithm36.Our analyses showed both a linear and a quadratic relationship between LEAPD indices and MoCA scores. However, the quadratic relationship was stronger, suggesting a complex relationship between cortical neurophysiology and cognition. The bounded non-gaussian feature of LEAPD scores, combined with this predominantly nonlinear, albeit monotonic, relationship with MoCA scores, made Spearman’s rho a more robust measure of correlations compared to Pearson’s38 and that our approach is sensitive to cognitive function across different domains. Although various other measures have been used to assess cognitive impairment via EEG40, to the best of our knowledge no other technique has this level of accuracy and correlation in PD patients and is as computationally-efficient. While LEAPD was useful in both PD and demographically similar control participants, its lower performance with control participants might be due to the limited range of performance in this healthy group with a small number of cognitively impaired participants. At its core, LEAPD is a data-driven method originally designed for PD vs control classification that captures characteristic spectral changes and can differentiate between data population groups such as PD and control groups or PD with and without depression with high accuracy31. In this study, LEAPD was able to provide cognitive markers in both PD and control participants by rapidly capturing the EEG spectral profiles while showing no significant effects of PD in the detection of cognitive impairment. This general effectiveness of LEAPD indices might lead to early diagnosis of cognitive impairment in not only PD but also other brain diseases that impair cognition that manifests through EEG such as Dementia with Lewy bodies (DLB)21.Strong correlations of LEAPD-based cognitive indices with PVT, DCCST, PCPST, PSMT, and FICAT scores in addition to MoCA suggest that cognitive impairment involves dysfunction in cortical networks that manifest in scalp EEG41. Furthermore, changes in EEG activities related to cognitive function can occur synchronously over multiple spectral rhythms. For example, delta-alpha coupling correlates with cognitive measures25, and cue-related medial frontal delta and theta power correlate with cognitive function in PD42. Abnormal delta and alpha rhythms in the posterior brain regions can index the decline of cognitive visuo-spatial function38. Delta, theta, alpha, and beta activities in EEG and their ratios reflect cognitive function in PD43. In our study, MoCA correlations of alpha-theta ratio and beta power in specific regions indicated synchronous changes in EEG over a broad spectrum. While changes in multiple EEG rhythms are traditionally captured with spectral ratios, which can correlate with cognitive measures in PD29, these ratios are limited by specific choices of spectral rhythms. In contrast, LEAPD is not strictly confined to the traditional spectral rhythms and captures simultaneous changes in multiple EEG rhythms by extracting the spectral profile in a holistic fashion, which led to an efficient estimation of cognitive dysfunction in this study. In line with previous studies, our results also indicated that the neurophysiological changes related to cognitive fluctuation are encoded mostly in spectral features rather than intra-channel connectivity measures20. Furthermore, the LPC models in LEAPD can reveal changes in the underlying oscillatory modes of the EEG signal. In our study, the dominant theta-alpha and beta oscillations captured by LEAPD at the optimal central-parietal electrodes indicated gradual shifts with cognitive function. Interestingly, spectral peak shifts of theta-alpha EEG rhythms in posterior cortical regions can predict dementia development in PD44. Shifts of peak frequency from alpha to theta with cognition, as discovered by LEAPD  and thus could effectively identify the characteristic cognitive fluctuations of DLB or PD dementia39. Complementary strengths of LEAPD to neuropsychological testing include speed, repeatability, and independence from patient-related factors such as fatigue and inability to participate due to severe motor symptoms or dementia. Future longitudinal studies are needed to explore these aspects. Furthermore, combined with advanced therapies such as adaptive neurostimulation with long-term wireless streaming of neural recordings47, EEG-based assessment of cognitive function by LEAPD might help design better feedback signals or select appropriate neurostimulation settings in targeted treatments for mitigating stimulation-related cognitive side effects and identify patients at risk in future studies9.EEG-based cognitive marker provided by LEAPD might be a useful complement to classical neuropsychological tests by choosing candidates as a large-scale screening and follow-up tool for further detailed testing leading to a more feasible utilization of such valuable resources and trained health professionals that are sometimes not immediately available21. Second, MoCA-based classification (cut-off 26/30) for cognitive impairment may not be as accurate as classification using Level II testing for cognitive impairment in PD5. However, our primary goal of this study was to index cognition using EEG that strongly correlates with standard measures of cognition. Third, our study is cross-sectional and represents a moderately sized convenience sample. Longitudinal studies with larger cohorts with repeated measurements are needed to determine the predictive value of our EEG-based markers on cognitive outcomes and their usefulness in monitoring cognition in PD in clinical practice. Fourth, our study was ON levodopa as usual, although our past work indicates that levodopa does not change EEG spectra, and our previous work showing levodopa differences was in rodent striatal field potentials, which are vastly different than scalp EEG48. Lastly, we had a moderate-sized dataset and thus evaluated the performance of LEAPD scores using cross-validation schemes, commonly utilized in measuring diagnostic performances50. Furthermore, we implemented subject-size cross-validation technique, the proper way of estimating a model’s performance in a diagnostic scenario50 with various values in k-folds, and repeated the process 100 times with independent shuffling of the folds. Performance variations of LEAPD indices across the cross-validation schemes were low  and robustness analyses showed stable performance. In addition, we trained LEAPD on data with randomly shuffled cognitive scores among subjects which showed no statistically significant correlation with cognitive scores or detection of cognitive impairment suggesting that the correlations with cognitive measures are not achieved through fitting noise or data artifacts.Our study has several limitations. First, scalp EEG has poor spatial resolution whereas MEG, fMRI, or intraoperative recording studies might provide detailed spatial information about brain activity, which could contribute to more accurate cognitive measurements of LEAPD. Despite the poor spatial resolution, we chose EEG as it is widely available, low-cost, and has shown promising results as a neurophysiological marker compared to others including fMRI, SPECT, PET, and neurophysiological tests in neurological conditions such as DLBIn summary, we found that LEAPD can provide a data-driven feature from resting-state EEG for cognitive measures in PD. This could be useful as a potential marker to infer cognitive impairment in PD or other neurological diseases. Because LEAPD is scalable and amenable to real-time applications, it also might inspire novel feedback-based interventions or advanced neuromodulation therapies targeted at cognition.


The vascular anatomy of the deep inferior epigastric artery perforator (DIEP) flap has been well studied in the planning for autologous breast reconstruction. Preoperative imaging with computed tomography angiography (CTA) provides accurate assessment of this vascular anatomy, which varies widely across patients. Several papers to date have described their encounter with an anomalous “epiperitoneal” or “peritoneo-cutaneous” perforator during flap harvest, a perforator that pierces the posterior rectus sheath from a peritoneal origin, to traverse rectus abdominis and supply the DIEP flap integument. In the course of over 3,000 CTA assessments of the vascular anatomy of the abdominal wall, we have encountered dominant peritoneo-cutaneous perforators in 1% of cases, and smaller perforators seen in many more cases, approaching 5% of cases. With increasing sensitivity of imaging, we also describe a unique case of multiple large bilateral peritoneo-cutaneous perforators, and present these findings in the context of DIEP flap harvest. It is critical to recognize these peritoneo-cutaneous perforators preoperatively to avoid mistaking them for a DIEP during the raising of a DIEP flap. The routine use of preoperative CTA enables the safe identification of individual vascular anatomy, including significant peritoneo-cutaneous perforators. As a mainstay of autologous breast reconstruction, the deep inferior epigastric artery (DIEA) perforator (DIEP) flap and its vascular anatomy have been well studied. The DIEA, originating in the majority of cases from the external iliac artery, provides the blood supply to the skin and soft tissues of the lower abdominal wall.Several papers to date have described their encounter with an anomalous “epiperitoneal” or “peritoneo-cutaneous” perforator during the raising of a DIEP flap for breast reconstruction,Lasso et alInstitutional human research ethics committee approval was achieved , and full informed patient consent was undertaken. We thus describe the case of a 48-year-old female who underwent abdominal wall CTA as part of routine preoperative workup for DIEP flap breast reconstruction. Preoperative analysis of CTA images revealed the presence of multiple bilateral peritoneo-cutaneous perforators, a unique finding, as described above. In the course of over 3,000 CTA assessments of the vascular anatomy of the abdominal wall, we have encountered dominant peritoneo-cutaneous perforators in 1% of cases, matching the findings of Whitaker et al. However, smaller such perforators have been seen in many more cases than this, approaching 5% of cases. These perforators share some anatomical features, highlighted inIn our case of multiple bilateral peritoneo-cutaneous perforators, it was found that all perforators identified were periumbilical and shared no anastomoses with the DIEA perforator system . The DIPeritoneo-cutaneous perforators are an uncommon anatomical variant in the arterial supply of the abdominal wall. Significantly sized peritoneo-cutaneous perforators are thought to occur in about 1% of patients,Innumerous small peritoneal branches from the entire DIEA provide supply to the parietal peritoneum concordant with its cutaneous territory.Preoperative CTA has already been proven to be a useful adjunct by reducing operative times and allowing safer intraoperative flap dissection.


Cirrhotic cardiomyopathy is a syndrome of blunted cardiac systolic and diastolic function in patients with cirrhosis. However, the mechanisms remain incompletely known. Since contractility and relaxation depend on cardiomyocyte calcium transients, any factors that impact cardiac contractile and relaxation functions act eventually through calcium transients. In addition, calcium transients play an important role in cardiac arrhythmias. The present review summarizes the calcium handling system and its role in cardiac function in cirrhotic cardiomyopathy and its mechanisms. The calcium handling system includes calcium channels on the sarcolemmal plasma membrane of cardiomyocytes, the intracellular calcium-regulatory apparatus, and pertinent proteins in the cytosol. L-type calcium channels, the main calcium channel in the plasma membrane of cardiomyocytes, are decreased in the cirrhotic heart, and the calcium current is decreased during the action potential both at baseline and under stimulation of beta-adrenergic receptors, which reduces the signal to calcium-induced calcium release. The study of sarcomere length fluctuations and calcium transients demonstrated that calcium leakage exists in cirrhotic cardiomyocytes, which decreases the amount of calcium storage in the sarcoplasmic reticulum (SR). The decreased storage of calcium in the SR underlies the reduced calcium released from the SR, which results in decreased cardiac contractility. Based on studies of heart failure with non-cirrhotic cardiomyopathy, it is believed that the calcium leakage is due to the destabilization of interdomain interactions (dispersion) of ryanodine receptors (RyRs). A similar dispersion of RyRs may also play an important role in reduced contractility. Multiple defects in calcium handling thus contribute to the pathogenesis of cirrhotic cardiomyopathy. Cardiovascular abnormalities in cirrhosis include hyperdynamic circulation and cirrhotic cardiomyopathy (CCM); the former is the combination of decreased peripheral vascular resistance and increased cardiac output whereas the latter is a syndrome of cardiac contractile dysfunction in the absence of a primary cardiac condition ,2,3. TheThe pathophysiology of the cardiovascular system is complex. In cirrhotic patients and animal models, the abnormal architecture of the liver causes portal venous hypertension, and the congested gut results in increased intestinal permeability. These pathophysiological changes trigger bacterial overgrowth, translocation, and increased bacteria-derived products in their circulation, including endotoxins that elevate systemic pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNFα), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Thus, patients with cirrhosis and portal hypertension manifest an inflammatory phenotype that leads to increased vasodilators such as nitric oxide, carbon monoxide, prostaglandins, bile acids, and glucagon. Therefore, the peripheral vasculature is dilated.It is well noted that the sympathetic nervous system (SNS) tone is increased in cirrhotic patients (serum concentration of norepinephrine is increased) . The incThere are numerous pathogenic mechanisms underlying CCM which broadly comprise two distinct pathways: factors related to (a) liver dysfunction with synthetic failure/dysregulation of substances such as proteins, lipids, lectins, hormones, etc., and (b) portal hypertension with gut congestion and induction of the inflammatory phenotype. A detailed discussion of these mechanisms is beyond the scope of this current review on calcium dysregulation , leading to calcium release from the sarcoplasmic reticulum (SR). The released calcium binds troponin C, which enables the movement of tropomyosin and thus exposes the myosin binding site on the thin filaments  20]. A . A 20]. At phase 2 of the action potential, calcium enters the cytosol through L-type calcium channels which activate ryanodine receptors . Activation of ryanodine receptors triggers the release of calcium from the sarcoplasmic reticulum. Calcium released mainly from the sarcoplasmic reticulum combines with the troponin complex and triggers actin–myosin cross-bridge linking, and thus cell contraction. This process is called excitation–contraction coupling.Calcium is an essential second messenger in all living organisms. From Caenorhabditis elegans to mammals, calcium is mandatory for body functions, locomotion, and neural activities . In the p < 0.03) as was the current of L-type calcium channels . Our study [p < 0.0001) [The abundance and current of L-type calcium channels are decreased following the development of cardiac dysfunction. Mukherjee et al.  studied ur study  on cirrh 0.0001) . At all The response of peak inward calcium current to maximal isoproterenol stimulation was also significantly lower. Protein expression and messenger RNA transcription for RyR2, SERCA2, and calsequestrin were quantitatively unchanged .Ryania speciosa plant used by South American Indigenous people in blow darts to paralyze prey. When ryanodine binds RyR channels, they induce an open state which results in an uncontrolled release of calcium from the SR, leading to muscle tetany. The flux of cytosolic calcium transients, calcium influx, and efflux should be balanced to maintain normal contractile function. RyR dysfunction is associated with a variety of conditions such as cardiac arrhythmias and heart failure. Although there are no structural changes in the intracellular calcium handling system in cirrhotic cardiomyopathy [It is now clear that the density and function of L-type calcium channels are reduced in cardiomyocytes from cirrhotic rats. This abnormality impacts the calcium transients in the intracellular calcium handling system, mainly ryanodine receptors (RyRs). RyR is a homotetrameric intracellular calcium release channel that is composed of four 565-kDa protomers and is located on the SR in cardiomyocytes and other cell types . The nammyopathy , the intThe functional unit  is composed of the L-type calcium channel and ryanodine receptor clusters (RyR clusters) which play an essential role in regulating calcium transients. In an in vivo study, Kolstad and co-workers  showed tIt is well known that the sympathetic nervous system is over-activated in patients with cirrhosis. In the early 1960s, Shaldon et al.  measuredThe ventricular systolic force depends on the magnitude of the cytosolic calcium transient and the sensitivity of the contractile proteins to the calcium. We showed that cellular calcium transients are reduced in cardiomyocytes from BDL-cirrhotic rats, which explains the decreased systolic force in these animals . AnotherTitin, a giant muscle protein, is mainly involved in ventricular diastolic function. In effect, its ‘spring-like’ elasticity is responsible for much of the passive mechanical recoil of early diastole. Kellermayer et al. demonstrated that titin abnormalities are associated with dilated cardiomyopathy. Our group did not find any direct titin structural abnormalities in cirrhotic rat hearts. However, we showed that a titin modulator, protein kinase A, is decreased, which may contribute to abnormal titin function and thus reduced diastolic compliance in the cirrhotic ventricle .SL) to evaluate the amount of spontaneous sarcomere length fluctuation during diastole and found that RMSSL is significantly higher in ventricular trabeculae from cirrhotic rat hearts at all stimulus rates compared with that from sham-control rats [Another abnormality of the intracellular calcium handling system is the calcium leakage from the SR. Calcium leakage reduces cardiac contractility in heart failure . We examrol rats  , a calcium-independent transient outward K+ current, and Isus, a delayed-rectifier K+ current. These abnormalities may lengthen the action potential duration of cardiomyocytes from the cirrhotic heart. The prolonged action potential duration decreases the efficiency of SR calcium release and therefore prolongs cardiomyocyte contraction/relaxation. Sah et al. [2+ transient in our cirrhotic cardiomyocytes may be responsible for slowed contraction in BDL hearts. We further found that the maximum calcium transient is significantly smaller in cardiomyocytes from cirrhotic hearts compared with that from sham controls. Time-to-peak calcium transient in cardiomyocytes from cirrhotic hearts is significantly longer than that from sham controls . We also demonstrated increased iNOS [1-AR [Shao et al.  evaluatesed iNOS  and decrOS [1-AR ,48 in ciOur lab  recentlyOur previous studies demonstrated the role of TNFα in cirrhotic cardiomyopathy ,49. We dSeveral studies demonstrated that TNFα modulates the expression of calcium regulatory proteins. Kao et al.  incubateIn rats with cirrhosis induced by bile duct ligation, oxidative stress is increased, which causes cardiac dysfunction ,15. HoweIt is now clear that abnormalities of the calcium handling system play an essential role in cirrhotic cardiomyopathy. In addition to L-type calcium channels, RyR2 dispersion may also be a culprit in the pathogenesis of cardiac dysfunction in cirrhosis. Any agents that prevent RyR2 dispersion or restore RyR2 integrity of interdomain interactions may exert beneficial therapeutic effects on cirrhotic cardiomyopathy.Oxidative stress affecting RyR2 destabilizes interdomain interactions within the RyR2 (RyR2 dispersion) which causes calcium leakage from the SR and seems to play a key role in the pathogenesis of heart failure . The antβ-blockers are commonly used for the treatment of cardiac tachyarrhythmias, high blood pressure, and congestive heart failure. β-blockers are also administered to patients with cirrhosis, mainly for gastrointestinal bleeding . Shen etAnother possible therapeutic strategy is targeting bile salts. Desai et al.  analyzedIn conclusion, the calcium handling system plays a key role in non-cirrhotic and cirrhotic cardiovascular diseases. Many etiologic factors that play important roles in cirrhotic cardiomyopathy also impact the calcium handling system, such as TNFα and oxidative stress. The factors that inhibit cardiac contractility in cirrhotic cardiomyopathy may act by impacting the cardiomyocyte calcium handling system.


Takotsubo cardiomyopathy (TCM) is a sudden, transient myocardial stunning precipitated by severe emotional or physical stress. It is characterized by left ventricular apical ballooning and elevated cardiac enzymes without significant coronary artery stenosis. Stress-induced catecholamine surge has been proposed to be the likely mechanism of TCM. We report the case of a 23-year-old female who presented to the emergency department unconscious and in respiratory distress following a motor vehicle accident. The point-of-care ultrasonography showed prominent B lines in bilateral lung fields and a dilated inferior vena cava (IVC). An x-ray and computed tomography (CT) scan of the chest revealed bilateral diffuse ground glass opacities. A CT scan of the brain showed a subarachnoid hemorrhage (SAH). Electrocardiography (ECG) showed normal sinus rhythm, but troponin I was elevated. Echocardiography revealed left ventricular apical hypokinesia. The coronary angiogram was normal. A diagnosis of TCM with SAH was made. Appropriate emergent care was provided, and at follow-up, she made a complete cardiologic recovery. TCM is a puzzling condition in an emergency setting and accurate and timely diagnosis is imperative in the management. Early prevention of hypoxemia and maintenance of mean arterial pressure and cerebral perfusion pressure is critical in determining the long-term outcome of the patient in the setting of co-existing CNS pathologies. Takotsubo cardiomyopathy (TCM) is a sudden, transient myocardial stunning characterized by apical ballooning of the left ventricle (LV) with no significant coronary artery stenosis. It was first described by Sato et al. in Japan in 1990. Patients often present with chest pain, elevated cardiac enzymes, and electrocardiography (ECG) changes typical of an acute coronary syndrome (ACS). The syndrome is often precipitated by severe emotional or physical stress [The diagnosis of TCM can be made using the modified Mayo Clinic criteria, which require the presence of regional wall motion abnormalities (RWMAs) of the left ventricle extending beyond a single epicardial vessel distribution, the absence of coronary artery obstruction or acute plaque rupture in angiography, new ECG abnormalities or elevation of cardiac enzymes, and the absence of myocarditis or pheochromocytoma .The exact pathophysiology of TCM still needs to be completely understood. Stress-induced catecholamine surge has been proposed to be the likely reason for TCM . MicrovaHypotension and cardiogenic pulmonary edema are the most common immediate complications of TCM due to acute LV dysfunction. Dysrhythmias, mitral regurgitation, LV thrombus formation, LV rupture, and death can also occur in the acute setting . TCM is 2) of 60%. Computed tomography (CT) of the chest  with a history of a motor vehicle accident. On arrival in the ED, the patient was in an altered sensorium with a Glasgow Coma Scale (GCS) score of E2V3M5. She was in respiratory distress, with a respiratory rate of 36/minute and an oxygen saturation of 75% on room air. Her heart rate was 126 beats per minute, and her blood pressure (BP) was initially 100/70 mmHg. Two large-bore intravenous cannulas were secured, and intravenous fluids were initiated. The patient was intubated with an 8.0 endotracheal tube, fixed at 20 centimeters (cm) from the incisors, and put on mechanical ventilation in volume-controlled ventilation (VCV) mode. Her saturation improved to 90%. Pink froth was coming through the endotracheal tube, suggestive of pulmonary edema. Extended focused assessment with sonography in trauma (eFAST) study showed no free fluid in pleural, pericardial, or peritoneal cavities, but there were prominent B lines in bilateral lung fields in all four zones of both lungs. The inferior vena cava (IVC) measured 2.1 centimeters in diameter with no variation in respiration , or cardiogenic pulmonary edema . A thoroHypotension unresponsive to intravenous fluid therapy and worsening pulmonary edema point to a cardiac pathology as the cause for the acute clinical presentation. An echocardiogram showed LV apical ballooning and hypokinesia with basal hyperkinesia, typical of TCM. The appearance of the heart is often compared to that of a Japanese octopus pot Figure . SpecifiTCM generally presents as an acute cardiovascular compromise characterized by severe left ventricular dysfunction. TCM, however, differs from an ACS because patients generally have a normal coronary angiogram. The left ventricular dysfunction extending beyond the territory subtended by a single coronary artery recovers within a few days or weeks . It is uTCM is one of the most frequently encountered cardiac debilities precipitated by CNS disorders like subarachnoid hemorrhage . SAH canThe apical ballooning in TCM can be explained by the regional differences in the density of β-adrenoreceptors in the heart. β2-adrenoceptors are more frequently expressed in the apical than in the basal segments of the LV, whereas β1-adrenoceptors are expressed much more at the base than at the apex of the LV . SupraphThe clinical course of TCM is typically self-limiting, with complete recovery within weeks . EmergenTCM is a puzzling condition in an emergency setting, and physicians might fall short of identifying this unique diagnosis, especially in this age group. An accurate and timely diagnosis is imperative, as the management is substantially different from other possible differential diagnoses. Unwarranted use of fluids and blood products to correct hypotension in a patient with NSM caused by trauma can be catastrophic. Maintenance of mean arterial pressure to prevent hypoxemia and to maintain cerebral perfusion pressure is challenging and critical in TCM/NSM in coexisting CNS pathologies like SAH. Other alternative intrathoracic diagnoses like pneumothorax, cardiac contusion, aortic injury, pericardial effusion, pulmonary contusion, acute coronary syndrome, or other primary cardiac pathologies causing pulmonary edema must be ruled out.


The majority of populations in developing countries are living in areas of no access or limited access to prehospital emergency medical services (EMS). In Addis Ababa, the reported prehospital EMS utilization were ranging from zero to thirty-eight percent. However, there is limited research on reasons for the low utilization of prehospital resources in Ethiopia. This study aimed to assess factors associated with prehospital EMS utilization among critically ill COVID-19 patients in Addis Ababa, Ethiopia. A hospital-based cross-sectional study was conducted to collect primary data from 421 COVID-19 patients in Addis Ababa between May and July 2021. Logistic regression was used to identify factors associated with prehospital service utilization. Andersen’s Behavioral Model was implemented to address independent variables, including predisposing, enabling, need, and health behaviors-related variables. The level of prehospital care utilization was 87.6%. Being married [AOR 2.6], belief that self-transport is quicker than the ambulance [AOR 0.13], and perceptions that ambulance provides transportation service only [AOR 0.14] were predisposing factors associated with prehospital service utilization while the source of referrals [AOR 6.9], and prior knowledge on the availability of toll-free ambulance calling numbers [AOR 0.14] were identified as enabling factors. Substantial proportions of critically ill COVID-19 patients used prehospital services to access treatment centers. Prehospital EMS utilization in this study varies by predisposing and enabling factors, particularly: marital status, source of referral, prior knowledge on the availability of toll-free ambulances, belief that self-transport is quicker than ambulances, and perceptions that ambulance provides transportation service only. Our findings call for further actions to be taken by policymakers including physical and media campaigns focusing on the identified factors. There is a growing need for prehospital emergency services worldwide as a result of the global increase in the emerging and evolving burden of diseases that require emergency care systems for prehospital treatment and/or transport to a hospital for further treatment –4. SimilSevere infection with COVID-19 can induce a life-threatening condition such as acute respiratory distress syndrome (ARDS) –10, whicAs of January 26, 2022, a total of 463,047 confirmed cases and 7,280 deaths from COVID-19 were reported to the World Health Organization (WHO) from Ethiopia . Implying that, the patients referred from public health care facilities were 6.9 times more likely to use prehospital EMS compared to self-referral patients. In addition, the patients who had no awareness of the availability of toll-free ambulance calling numbers were 86% times less likely to use prehospital care services compared to others [AOR 0.14] .A statistically significant association was not observed for need factors on multivariate logistic regression. However, having at least one chronic comorbid illness [COR 2.0]; known diabetes [COR 2.4] and a history of hypertension [COR 2.3] were associated with prehospital care service utilization without adjusting for potential confounders .The importance of prehospital care use is centered on its capacity to increase the chance of survival and reduce morbidity related to emergency medical illness , 27. HowMarital status was associated with prehospital care use in this study. Previous studies from Ethiopia and elsewhere seldom reported an association between marital status and prehospital care utilization , 32, 33.A previous study from Ethiopia reported that most of the patients that used ambulances were transported from health care facilities than the community . AlthougThe current study shows that prior knowledge of the existence of toll-free numbers to transport COVID-19 patients was independently associated with prehospital emergency medical service use. In agreement with our finding, a previous study from North Ethiopia reported an association between ambulance use and prior knowledge of ambulance calling numbers among laboring women .Although a statistically significant association was not established on adjusted analysis in this study we have observed a significant association between respondents’ awareness of the availability of free ambulance services to transport COVID-19 patients and prehospital services utilization on unadjusted analysis. The study from Mekele city, the capital of Tigray regional state of Ethiopia, evidenced an association between ambulance use and participants’ prior knowledge of the availability of charge-free prehospital services for laboring mothers . KnowledOne of the limitations of this study is that the respondents were recruited only from public COVID-19 treatment centers. Patients treated at private COVID-19 treatment centers were not included in the study and this may limit the generalizability of our findings. Although it could be one potential enabling factor, the variable monthly income was removed from the analysis because more than half of the respondents resisted disclosing their monthly income and this may affect our findings. Lastly, it was a cross-sectional study with the usual inherent limitations. Therefore, the generalization of our findings should be made considering the mentioned limitations.Despite the aforementioned limitations, this study was conducted in a multicenter, guided by a model to address explanatory variables, and focused on less researched aspects of COVID-19 research .We identified that about nine in ten seriously ill COVID-19 patients used prehospital emergency medical services to access treatment centers. EMS use in this study varies by predisposing and enabling factors. The identified predisposing factors were marital status, belief that self-transport is quicker than ambulance, and perception that ambulance crew provides transport service only. Meanwhile, the identified enabling variables were a source of referrals and prior knowledge on the availability of toll-free numbers to call ambulances. Our findings call for further actions to be taken by policymakers including physical and media campaigns addressing the identified factors.S1 Data(SAV)Click here for additional data file.


Mean size and gyrification measures were extracted using FreeSurfer software for each cortical region of interest. FreeSurfer software was also used to generate subcortical volumes for regions in the automatic subcortical segmentation. For cortical analyses, separate ANOVA analyses of size  and gyrification  measures were conducted to test for a main effect of diagnosis  and the interaction of diagnosis-group with age-group  across cortical regions. Cortical analyses were first conducted across a set of regions that comprise the speech network and then in a second whole-brain analysis. Next, separate ANOVA analyses of volume were conducted across subcortical regions in each hemisphere. False discovery rate corrections were applied for all analyses. Additionally, we tested for correlations between structural morphometry and stuttering severity. Analyses revealed thinner cortex in children who stutter compared with controls in several key speech-planning regions, with significant correlations between cortical thickness and stuttering severity. These differences in cortical size were not present in adults who stutter, who instead showed reduced gyrification in the right inferior frontal gyrus. Findings suggest that early cortical anomalies in key speech planning regions may be associated with stuttering onset. Persistent stuttering into adulthood may result from network-level dysfunction instead of focal differences in cortical morphometry. Adults who stutter may also have a more heterogeneous neural presentation than children who stutter due to their unique lived experiences.This cross-sectional study aimed to differentiate earlier occurring neuroanatomical differences that may reflect core deficits in stuttering versus changes associated with a longer duration of stuttering by analysing structural morphometry in a large sample of children and adults who stutter and age-matched controls. Whole-brain T et al. analyse cortical morphometry in both children and adults with persistent developmental stuttering and identify differences in cortical thickness in several key speech planning regions, particularly in children, with significant correlations between cortical thickness and stuttering severity in these brain regions.In this work, Miller   Stuttering occurs in up to 10% of pre-school children,6-11 and decreased activation during speech in left sensorimotor regions.12 Neuroimaging studies of children who stutter (CWS) also suggest differences in brain structure and function in the left hemisphere speech network, including left IFG and premotor regions.13-16Neuroimaging studies of adults who stutter (AWS) reveal aberrant structural and functional connectivity in the left hemisphere speech network, particularly in the left inferior frontal gyrus (IFG) and ventral premotor cortex (vPMC),17-24 One source of this inconsistency is differences in the measures used to quantify cortical morphometry. Historically, volume-based methods like voxel-based morphometry measured the relative volumes of grey matter and white matter. Increasingly, surface-based methods allow for differentiation of cortical thickness and surface area, as well as measurement of cortical gyrification. Local gyrification index (LGI) provides a measure of the proportion of cortex located within a sulcus compared with the outer cortical surface and is calculated within circular regions surrounding each vertex on the cortical surface.26 These measures of grey matter morphometry provide improved insight into the precise neuroanatomical differences that underlie differences in neural processing.27 Cortical thickness changes are believed to reflect changes in the number and arrangement of neurons within cortical columns as a result of dendritic arborization, pruning, and myelination, which improve the efficiency with which neural signals are transmitted,28-30 while surface area instead measures the number of cortical columns.30 Cortical gyrification has been shown to reflect the neural circuitry within and between brain regions and supports the expansion of surface area across the brain.32Although some consensus is emerging regarding potential brain regions involved in stuttering, the precise differences in cortical structure responsible for stuttering remain unclear. Structural morphometric studies in AWS have been largely inconsistent with little overlap between reported locations of structural abnormalities.Another key difficulty in stuttering neuroimaging research is differentiating the primary deficits responsible for stuttering from secondary compensatory responses developed in response to years of dysfluent speech. Differences in brain anatomy and function in AWS may represent the underlying neuropathology responsible for speech dysfluency or may instead develop in response to secondary compensatory mechanisms that cause changes in brain anatomy and function. This confound complicates the interpretation of many neuroimaging studies, which have often included only adults. Although some degree of compensatory change may also be present in CWS given the significant degree of neural plasticity in childhood, differences in brain structure in CWS are more likely to reflect the core neuroanatomy underlying the disorder, given the shorter duration of stuttering.34 New methods in data harmonization, such as the ComBat tool,35 attempt to control for scanner-related variation across sites to ensure valid findings in multi-site studies.A further limitation of prior imaging work in this field is the relatively small sample sizes. Additionally, methodological differences across studies limit direct comparison between CWS and AWS from prior studies that separately studied these two age-groups. Multi-site neuroimaging studies allow for the application of standardized analyses across larger datasets for more robust neuroimaging analyses, given the significant time and cost of collecting this data. However, differences across MRI scanners have the potential to introduce unwanted variance into measures of grey matter morphometry.The current study extends prior cortical morphometry studies through direct comparison of cortical morphometry in a sample of both CWS and AWS pooled across eight prior studies. This study aims to: (i) determine the primary neuroanatomical correlates of stuttering in both children and adults in the largest neuroimaging sample of individuals with persistent developmental stuttering to date, (ii) confirm prior findings of secondary neural differences present only in CWS or AWS, and (iii) identify regions-of-interest (ROIs) where structural morphometric measures are a significant predictor of stuttering severity.36-42 consisting of 92 AWS , 99 adults with no stutter , 74 CWS  and 92 children with no stutter . We will use persons who stutter (PWS) or persons with no stutter (PNS) to refer to the combined child and adult groups, while CWS and AWS will refer to the specific age-group.This cross-sectional study pooled data from 357 unique participants across 8 previous stuttering studies,37All participants were American English speakers with no contraindications for MRI or a history of speech or language disorders. Diagnosis was confirmed by a speech-language pathologist for all PWS, as part of the inclusion criteria for the original studies. Further, inclusion criteria for CWS in the current study included classification as ‘persistent’, over the course of a 4-year longitudinal study . Based on these criteria, we excluded 23 participants who were considered recovered at the end of this longitudinal period.t-test: t(355) = −1.31, P = 0.19], sex  and handedness . Within the child age-group, CWS and CNS groups were also matched for non-verbal IQ . Participants provided written informed consent or assent, depending on age, in accordance with the human subjects policies in place at each institution.All PNS participants reported no diagnosis or history of stuttering. Additional inclusion criteria for CNS included no family history, no parent concern regarding child’s fluency, and <3% stuttering-like disfluencies in a speech sample. PWS and PNS groups were well matched for age .SSI-4 scores for AWS ranged from 6 to 48 [mean: 23.9 (SD: 8.8)], representing a wide range of symptom severity. SSI-4 scores were not available for one AWS who was not included in SSI-4 analyses. Severity in CWS ranged from 8 to 48 [mean: 19.8 (SD: 6.7)]. Severity in CWS was significantly lower than in AWS . Therefore, all covariates were included in subsequent models. Both cortical thickness and LGI decreased with age. Female subjects exhibited decreased surface area compared with males. Surface area and LGI were both higher in left-handed participants.A multiple regression analysis identified significant covariation between the selected morphometric measures  and all four potential covariates . Post hoc analysis within this region revealed significant effects in both of the comprising ROIs: supplementary motor area  and pre-SMA .Gyrification analyses tested for either a significant main effect of stuttering diagnosis across the lifespan or a significant diagnosis by age-group interaction. Hierarchical analysis of LGI within the left hemisphere speech network revealed significant effects only in the medial premotor region , mediated by a significant stuttering by age-group interaction in both ROIs . The interaction was driven by a significant group difference only within the child age-group (CWS–CNS contrast) in both ROIs (P < 0.005), with CWS demonstrating higher gyrification (P > 0.30).fication . SeveritF = 5.30, P = 0.0003, p-FDR = 0.005], IFG, pars opercularis , posterior inferior frontal sulcus  and anterior insula . Within the opercular region, neither ROI demonstrated significant effects (p-FDR = 0.060). Within the triangularis region, significant effects were isolated to the right anterior frontal operculum . Significant ROIs identified in the speech network analyses are summarized in Post hoc analyses characterized the effects in right aFO, right pIFS and right aINS as follows:Replication of this hierarchical analysis in the right hemisphere speech network revealed significant effects in four functional ROIs: IFG, pars triangularis  and a significant interaction with age-group . Significant differences within age-groups were present only between AWS and ANS, with reduced gyrification in AWS  and significant main-effect of stuttering diagnosis , due to significant differences only within the child group . CWS displayed increased gyrification compared with peers  = 2.12, P = 0.035, p-FDR =0.046] and a significant main-effect of stuttering diagnosis , driven by differences only between AWS and ANS . Similar to the pattern seen in the right aFO .Neither ROI was correlated with stuttering severity  = −3.76, P = 0.0002; with increased LGI in CWS] were responsible for a significant diagnosis by age-group interaction  and a significant main effect of diagnosis .Left mid cingulate gyrus: Significant differences between CWS and CNS only  resulted in both a significant diagnosis by age-group interaction  and a significant main effect of diagnosis .Right posterior inferior temporal gyrus: Significant differences between CWS and CNS only . Therefore, size analyses focused on differences in cortical thickness.Hierarchical analyses were performed across measures of cortical size  to test for a significant main effect of stuttering diagnosis or a significant stuttering by age-group interaction across the 14 functional speech network ROIs . First, F = 5.99, P = 0.00001, p-FDR = 0.001], left lateral premotor cortex , left lateral motor cortex ; left IFG pars triangularis ; left frontal orbital cortex ; left IFG, pars opercularis ; and left posterior supramarginal gyrus . Post hoc tests within each of these functional ROIs confirmed significant effects in all of the corresponding anatomical sub-regions except for left ventral motor cortex . Complete findings are summarized in ANOVA analysis of cortical thickness in the left-hemisphere speech network identified significant effects in the following functional ROIs: left medial premotor cortex  and pSMG . Within higher-order somatosensory cortex, only right anterior supramarginal gyrus (aSMG) demonstrated significant effects in post hoc testing . Right hemisphere effects were further characterized as follows:This hierarchical analysis was then repeated across the 14 homologue speech network ROIs in the right hemisphere, revealing significant effects in two functional ROIs: higher-order somatosensory cortex  and a significant interaction  were identified, driven by a significant difference only within children , with thinner cortex in this region in CWS.aSMG: Both a significant main effect  and significant interaction  were again driven by a significant difference between CWS and CNS groups only , again revealing thinner cortex in CWS.pSMG: A significant main effect . Further post hoc analysis revealed this effect was due to differences within the child age-group only , with decreased right caudate volume observed in CWS compared with CNS. Volume in this region was not correlated with SSI-4 (P > 0.25).ANOVA analysis of subcortical volume revealed no significant effects (diagnosis main effect or diagnosis by age-group interaction) in left hemisphere regions  an accelerated initial period of cortical growth in early childhood, (ii) higher peak cortical thickness and (iii) a more protracted period of cortical thinning into their adolescence, consistent with our findings.70-72 Therefore, the widespread differences in cortical morphometry in CWS may reflect the genetic basis of stuttering. Prior work has identified relationships between gene mutations associated with stuttering and disruptions in grey-matter volume and functional connectivity in CWS;73-75 our findings overlap with a number of both speech and non-speech regions identified with high expression of GNPTG, a lysosomal trafficking gene associated with stuttering.73 These non-speech findings also concur with prior reports of differences in connectivity in CWS in default mode, fronto-parietal and dorsal attention networks.76Cortical development is also strongly influenced by genetics.78 This circuit is a key component of the DIVA and GODIVA neurocomputational models.56 Within the DIVA framework, stuttering can be explained by damage to the cortico-basal ganglia loop at three possible loci: (i) within the basal ganglia, (ii) axonal projections between subcortical structures and cerebral cortex and/or (iii) cortex.79 Specifically, our analyses revealed reduced cortical thickness in CWS across a network of key left frontal and premotor regions that are identified in the DIVA and GODIVA models of speech production,56 in addition to uncorrected differences in the right caudate, consistent with prior reports of atypical caudate anatomy in CWS.80 Prior subcortical analyses in CWS have also found differences in key regions in the cortico-basal ganglia loop, albeit in other regions including the putamen and thalamus;37 however, our group comparisons tested for differences across the volume of the entire structure for each region, and thus, results from this study cannot be directly compared with studies that have reported subcortical differences based on a voxel-wise comparison in narrower age groups.Many of the regions identified in our speech network analyses are part of the basal ganglia-thalamo-cortical motor circuit, believed to be central to the disrupted initiation of speech-motor programmes in stuttering.36—left midPMC, left vPMC, and left anterior central operculum (aCO)—with the novel finding that cortical thickness in left midPMC is correlated with stuttering severity. Reduced cortical thickness in left lateral premotor cortex may be related to an impaired ability to activate speech motor programmes in CWS.In the DIVA model, vPMC is the site of a ‘speech sound map’ that essentially ‘reads out’ optimized motor programmes representing well-learned speech sequences via projections to motor cortex, the cerebellum, and the basal ganglia motor loop. Our cortical thickness analyses revealed significant differences between CWS and CNS in three of the motor and premotor ROIs previously identified in CWS53 Thus, atypical development of either region may result in failed initiation or termination of speech due to difficulty detecting the precise sensorimotor and cognitive context for initiation.79 Our finding of reduced cortical thickness in left SMA and preSMA in CWS corroborates prior findings of reduced grey matter volume in these regions in CWS,82 and is further supported by the association between stuttering severity and cortical thickness in left preSMA.The DIVA and GODIVA models further specify key roles for SMA and preSMA within the basal ganglia motor loop: bilateral SMA is a key hub for speech initiation, while bilateral preSMA is responsible for representations of metrical timing for fluent speech production.53 This region is involved in motivation and emotional aspects of vocal expression, including voluntary initiation of vocalizations.83 LGI has been shown to decrease during skill acquisition,84 which may reflect increased synaptic pruning for more efficient neural circuitry and increased long-range connectivity.31 Thus, increased LGI in preSMA and SMA may reflect increased short-range connections at the expense of long-range white matter connections along the frontal aslant tract, which are critical for fluent speech.85-87 Structural and functional connectivity in left SMA are both atypical in CWS and AWS,88 and left SMA is overactive in AWS during moments of dysfluent speech.89 Combined, these findings suggest that the cortical differences present in CWS may instead persist in the form of abnormal function or connectivity in this region in AWS.The role of medial premotor regions in stuttering is further supported by our findings of increased gyrification in both SMA and preSMA in CWS, as well as in the neighbouring mid-cingulate gyrus. The cingulate motor area represents ‘the will to speak’ in the DIVA model and is heavily interconnected with SMA and preSMA.81 Our analyses also identified reduced cortical thickness in CWS in left FOC, while in AWS, cortical thickness in left FOC was positively correlated with severity. Left FOC is believed to have a compensatory role, such that it promotes speech fluency: increased brain activation, increased structural white matter integrity, and increased functional connectivity between left FOC and the cerebellum have all been associated with less severe stuttering or stuttering recovery.90-92Similarly, our finding of reduced cortical thickness in bilateral supramarginal gyrus in CWS is consistent with reported reduced grey matter volume and fractional anisotropy in left supramarginal gyrus in CWS.94 Specifically, Foundas et al.94 reported the presence of an additional diagonal sulcus or an extra gyrus dorso-lateral to the IFG pars triangularis in many AWS. Both sulci serve as boundaries in the parcellation of aFO and would thus result in an anterior shift moving the aFO parcellation further from the Sylvian fissure. The cortex adjacent to the Sylvian fissure has a characteristically high LGI due to the high degree of cortical folding and submerged cortex in this major sulcus; therefore, an anterior shift in the aFO parcellation would result in lower LGI for individuals with this anatomic difference consistent with our findings of reduced LGI in AWS. Reduced LGI in AWS may also reflect increased long-range connectivity, potentially due to compensatory recruitment of a right-hemisphere feedback system.7 Our finding that these differences in right-hemisphere gyrification were not present in CWS suggests this difference is not a core deficit responsible for stuttering, but rather a compensatory difference that may develop in response to many years of dysfluent speech.Our analyses showed reduced LGI in right aFO and right aINS in AWS, consistent with previous studies demonstrating atypical gyral and sulcal patterns along the right Sylvian fissure in AWS.21 while others report no group differences in this region.23 The current study improves on prior work through (i) a much larger sample size and (ii) implementation of more sensitive surface-based cortical morphometry analyses. Surface-based measures provide improved spatial resolution across gyral boundaries compared with voxel-based morphometry, which is limited by the overlap of grey and white matter or neighbouring gyri within a single voxel. Additionally, the use of the SpeechLabel parcellation scheme increases statistical power by providing finer-scale demarcation of key speech regions and reducing the number of statistical comparisons, particularly compared with a voxel-wise analysis. Combined, our findings provide evidence that differences in cortical size found between CWS and CNS are no longer reliably evident by adulthood.We were unable to replicate any prior findings of cortical size differences in AWS, in line with the broader pattern of poor replicability across prior studies of structural morphometry in AWS. For example, prior studies have reported both increased and decreased cortical size in AWS within left IFG,This study aimed to identify neuroanatomical differences present in CWS that may reflect core deficits in stuttering. However, since these children had begun to stutter before the time of scanning it is possible that their brains have already begun to exhibit some degree of compensatory changes in response to their disfluent speech. Nonetheless, given the relatively short duration of stuttering in our CWS sample, these findings are more likely to reflect a core neuroanatomical feature of the disorder, particularly compared with AWS. Further, a significant proportion of the CWS sample are pre-school-age children (42/74 aged 3–5 years), who are very close to stuttering onset. Additionally, documentation of eventual recovery or persistence status following longitudinal follow-up further improves the sensitivity of our analyses to detect neuroanatomical differences associated with stuttering in young children, compared with prior literature that has often reported on older children and adolescents.37 Further, there is a high degree of overlap in the children included in both this study and in previous work published by our lab;36 although many reported findings in the initial study were confirmed in this larger cohort, a true replication of these results in an independent cohort, is necessary. Nonetheless, the comparison with cortical morphometry in adults is novel and revealed that none of the reported differences in CWS, considered to be potential hallmarks of the disorder, persist in AWS.Notably, although this pooled dataset allows for analysis of the largest published sample of PWS to date, the included datasets present several limitations including imbalanced sample sizes between stuttering and control groups and variation in the age ranges within child and adult age-groups. Further, the included datasets have a gap in participants between ages 11 and 18 years, in order to focus on the neural presentation of stuttering in younger children who are closer to stuttering onset. As a cross-sectional study, these results provide only a snapshot of potential differences in cortical morphometry in younger children and AWS. Our findings suggest differences in the developmental trajectory in CWS; however, longitudinal analyses are necessary to better characterize these differences.95 This may explain the negative findings in our adult sample as well as inconsistent results across prior studies in AWS that all included much smaller sample sizes than the current study. However, this current study differs from the work by Marek et al.95 in that it investigates a clinical-control contrast with a more focused sample and measures selection, and behavioural measures appropriate to characterize the expected variability in the sample. All of these factors can be expected to increase the size and reliability of the brain-behavioural associations evaluated in this study.96One further limitation is our sample size. Although our sample size is considerably larger than prior work in this population, recent findings suggest that much larger sample sizes (i.e. thousands of subjects) may be necessary to achieve adequate power for replicable identification of reproducible individual brain-behaviour associations across the entire brain, as many such associations show relatively small effect sizes.97 We have minimized these effects through (i) inclusion of datasets that are matched for cases and controls within each scanner (see 35 which has been demonstrated to effectively control for inter-scanner effects to allow for combination of multi-site datasets. Given no significant differences in the proportion of stuttering and control participants scanned at each site, the impact of scanner variability on our primary analyses comparing stuttering and control participants should be minimal. However, as shown in While pooling across multiple scanners can be advantageous in order to increase a study’s sample size and allow its results to generalize beyond the specificities of each particular site’s sample pool or acquisition methods, it can also bring a number of challenges when dealing with the treatment of potential inter-site differences. Measurements across MRI scanners have been shown to be quite stable, but variance across sites due to differences in scanner hardware or scan sequences can nonetheless potentially introduce substantial bias into analyses.nner see , and (iiOur findings suggest that morphometric measures are most sensitive to group differences in childhood when they better capture genetic influences on early cortical development and differences in the developmental trajectory. The identified cortical differences in CWS across key speech planning regions are consistent with prior literature. While these differences in cortical size and gyrification may be involved in stuttering onset in children, none of these identified differences persisted across the lifespan. Thus, additional research is needed to further characterize the role of white matter connections within the cortico-basal ganglia network to identify commonalities in how this network is disrupted across children and adults who stutter.


Immunotherapy has made significant advances in the treatment of colorectal cancer (CRC), revolutionizing the therapeutic landscape and highlighting the indispensable role of the tumor immune microenvironment. However, some CRCs have shown poor response to immunotherapy, prompting investigation into the underlying reasons. It has been discovered that certain chemotherapeutic agents possess immune-stimulatory properties, including the induction of immunogenic cell death (ICD), the generation and processing of non-mutated neoantigens (NM-neoAgs), and the B cell follicle-driven T cell response. Based on these findings, the concept of inducing chemotherapy has been introduced, and the combination of inducing chemotherapy and immunotherapy has become a standard treatment option for certain cancers. Clinical trials have confirmed the feasibility and safety of this approach in CRC, offering a promising method for improving the efficacy of immunotherapy. Nevertheless, there are still many challenges and difficulties ahead, and further research is required to optimize its use. DespIt was previously believed that chemotherapy was solely an immunosuppressive agent. However, recent data indicate that chemotherapy drugs can promote immune activation through various pathways, notably via the induction of immunogenic cell death (ICD) mechanisms –9. Certa2Immune checkpoint inhibitors (ICIs) have brought new opportunities in cancer treatment –15. The Despite the tremendous potential of immunotherapy in CRC treatment, there are also challenges and limitations. CRC is a common malignancy, with the majority being MSS/pMMR type. Compared to MSI-H/dMMR tumors, MSS/pMMR tumors have poorer response to immunotherapy, primarily due to immune suppression or immune desertification –23, charV600E mutation has been shown to reduce T cells infiltration into the TME and eliminate neoantigen presentation on cancer cells ) and immune inhibitory molecules (such as IDO1 and transforming growth factor-beta (TGF-β)) are elevated –50. ThisIn MSS/pMMR CRC, inhibition of the tumor cell apoptosis pathway renders the tumor cells insensitive to attacks from the immune system. Aberrant activation of the WNT/β-catenin signaling pathway is frequently observed in MSS CRC but is rare in MSI-H CRC , 55. AbnIn summary, immunotherapy has had a profound impact on the traditional treatment of CRC, but it also poses challenges and limitations. Future research needs to further explore the combination of immunotherapy with conventional chemotherapy, overcome immune resistance mechanisms, and understand the influence of the TIME on the efficacy of immune therapy in order to enhance the effectiveness of immunotherapy and advance the treatment of CRC.3Recent research in the field of immunotherapy has demonstrated that chemotherapy drugs not only enhance the immunogenicity of cancer cells but also induce immune stimulation by activating effector T cells and inhibiting immune suppressive cells. These exciting findings suggest that the combination of chemotherapy and ICIs may have synergistic anti-cancer effects, providing a promising treatment option for tumor patients who have a poor response to monotherapy with ICIs.Studies have shown that chemotherapy drugs may exert their effects through immune stimulation mechanisms . For exaThe TIME is a key barrier to anti-tumor immunity and may limit the clinical efficacy of immunotherapy , 88. ImmFurthermore, some studies suggest that chemotherapy-induced DNA damage fragments in the cell nucleus may actively translocate to the cytoplasm to prevent their erroneous insertion into the genomic DNA. This process activates the innate immune cGAS-STING (cyclic GMP-AMP synthase/stimulator of interferon genes) pathway, leading to an immune-rich microenvironment and triggering innate immune responses –102.H cells) (FH and TH1 cells via the ICOSL-ICOS axis. The inducible T-cell co-stimulatory (ICOS) pathway is another important pathway in tumor immunotherapy. ICOS is a co-stimulatory receptor expressed on activated T cells and is crucial for their survival and function. The ICOS pathway plays a critical role in balancing effector T cells and Tregs, and its dysregulation has been associated with the development and progression of various types of cancer  and effector helper T cells  after treatment with Irinotecan  and 18-month PFS rate (28% vs. 9%), and ORR increased from 46% to 60%.FOLFOX plus bevacizumab is the first-line standard of care (SOC) for MSS mCRC. The Checkmate 9X8 study  challengThe BACCI phase II trial    is a sinA study  evaluateChemoradiotherapy also plays an important role in enhancing tumor response to immunotherapy. Current research indicates that radiation therapy can increase the expression of antigens on tumor cells, enhance tumor cell immunogenicity, and promote immune cell infiltration , 187. ThMost immunogenic chemotherapy agents have been shown to evoke immune stimulation not only by increasing the immunogenicity of cancer cells, but also by activating effector T cells and suppressing immune suppressor cells. These results suggest that the combination of chemotherapy and ICIs can have a synergistic anticancer effect and indicate that chemotherapy in combination with immunotherapy may be suitable for tumors that respond poorly to ICIs monotherapy. There are also ongoing prospective studies whose safety has been proven feasible, and the results of which are highly anticipated.6Considering the immune-activating effects of chemotherapy drugs, the combination of chemotherapy and ICIs is an appropriate partner to achieve rapid and long-term cancer control. Based on these findings, we propose the concept of inducing chemotherapy, which involves using immunogenic chemotherapy drugs to change the timing before immunotherapy, converting “cold” tumors into “hot” metastases to initiate or restore anti-tumor immune responses, thereby enhancing the efficacy of ICIs . Some prSong et al.  investigDosset et al. conducted an interesting preclinical study using a microsyngeneic mouse model of two MSI-H CRCs (CT26 and MC38) and observed that adding adjuvant ICIs after FOLFOX could induce complete and durable tumor responses, whereas FOLFOX or ICIs alone were ineffective . TherefoThe successful outcomes achieved in these preclinical trials provide strong evidence for the future implementation of clinical trials involving inducing chemotherapy Table 2.vs. 78.8%), with equivalent risks of late toxicities (≥grade 3) (11.3% vs. 11.4%). This study suggests that inducing chemotherapy before concurrent chemoradiotherapy can significantly improve the OS of patients with locally advanced nasopharyngeal carcinoma without increasing the risk of late toxicities.Ma et al.  presenteBased on the effective results obtained from preclinical trials, a series of clinical trials combining chemotherapy induction with immunotherapy have also been conducted in CRC, and significant progress has been achieved.in vivo, providing a basis for using these drugs to eliminate immune-suppressive cells and improve checkpoint efficacy in patients with metastatic MSS CRC. The combination of FTP/TPI and OXA has been shown to be safe and effective in a phase I human clinical trial , OXA, or combination therapy, especially TAM2, results in changes in the TAM1/TAM2 ratio, as well as enhanced infiltration and activation of cytotoxic CD8+ T cells, enhanced production of granzyme B, IFNγ, and TNFα in CD8+ T cells within the tumor, and upregulation of PD-L1 and PD-1 expression Figure 5al trial .A phase II clinical trial  is curreThe MAYA II phase clinical trial (NCT03832621)  studied A randomized phase II trial  evaluateA study  aimed toAlthough inducing chemotherapy combined with immunotherapy has achieved significant results, studies have shown that some chemotherapy drugs exhibit different immunogenic effects depending on their regimen, timing, dose, or administration sequence, even when used in combination with ICIs. One study investigated the impact of drug administration sequence and found that CTX given one day before anti-CTLA-4 therapy resulted in immune-mediated anti-tumor responses. However, when the sequence was reversed, CD8+ T cells underwent massive apoptosis, and the anti-tumor effect of anti-CTLA-4 was weakened . AnotherExcitingly, inducing chemotherapy has become one of the standard treatment options for certain tumors, and a series of clinical trials on inducing chemotherapy have been conducted in CRC, achieving promising results. We propose the exploration of personalized treatment plans, gradually reducing the chemotherapy regimen and combining it with immunotherapy after inducing chemotherapy, until only oral chemotherapy is maintained, and eventually achieving maintenance therapy with a single immunotherapy. We look forward to further confirmation and application in the future.7In the field of CRC treatment, the combination of inducing chemotherapy and immunotherapy has emerged as a promising therapeutic strategy. However, there are still challenges and directions that need to be addressed in the future.The TIME in CRC typically exhibits immunosuppressive features that limit the activity of immune cells. Future research can further explore the underlying mechanisms by which chemotherapy-induced immunotherapy overcomes this immune suppression, such as enhancing the activity of immune cells, modulating the polarization state of tumor-associated macrophages, disrupting tumor vasculature, and so on . To imprscRNA-seq technology provides us with an opportunity to gain in-depth understanding of tumor and immune cell heterogeneity , 210. ThDue to the limited predictive ability of current biomarkers such as PD-L1 expression and tumor mutation burden in cancer precision medicine, alternative biomarkers are still being explored , 216. A Furthermore, due to significant biological differences among CRC patients, personalized immunotherapy approaches become crucial. scRNA-seq may reveal potential mechanisms regulating immune cell exhaustion and identify advanced biomarkers, thereby facilitating the design of novel personalized immunotherapy strategies , 218–2208Immunotherapy has made significant progress in CRC, revolutionizing treatment outcomes. The TIME is closely related to tumor immunotherapy, which is a key obstacle to anti-tumor immunity and may limit the clinical benefits of immunotherapy. Recent studies have shown that some chemotherapy drugs can promote immune activation and enhance the efficacy of immunotherapy. By inducing ICD and exposing new antigens, it can activate CD8+ T cells and enhance the immune response to cancer. A large body of research has shown that most chemotherapy drugs exert immunostimulatory effects by inhibiting immune-suppressive cells or activating effector cells, or by increasing immunogenicity and T cells infiltration. inducing chemotherapy combined with immunotherapy has become a standard part of treatment in some tumors, and clinical trials have demonstrated its feasibility and safety in CRC. These combination therapies typically transform “cold” tumors that are insensitive to immune response into “hot” tumors. We propose a personalized exploration of inducing chemotherapy, gradually reducing chemotherapy regimens after systemic chemotherapy induction, combining with immunotherapy until only oral chemotherapy is maintained, and eventually transitioning to immune monotherapy maintenance treatment. In summary, chemotherapy-induced immunotherapy has enormous potential in the field of CRC. Future research will focus on overcoming the immune-suppressive microenvironment, applying scRNA-seq technology, achieving personalized treatment, researching predictive biomarkers, and optimizing combination therapy, among other challenges, to benefit more cancer patients in the near future.KJ and HL contributed to the conception and design of the study. SY drafted the initial manuscript and prepared the figures. YH and MY wrote sections of the manuscript. All authors participated in revising the manuscript, read, and approved the final version for submission.