Overcoming the H4K20me3 epigenetic barrier improves somatic cell nuclear transfer reprogramming efficiency in mice.

Epigenetic reprogramming during fertilization and somatic cell nuclear transfer (NT) is required for cell plasticity and competent development. Here, we characterize the epigenetic modification pattern of H4K20me3, a repressive histone signature in heterochromatin, during fertilization and NT reprogramming. Importantly, the dynamic H4K20me3 signature identified during preimplantation development in fertilized embryos differed from NT and parthenogenetic activation (PA) embryos. In fertilized embryos, only maternal pronuclei carried the canonical H4K20me3 peripheral nucleolar ring-like signature. H4K20me3 disappeared at the 2-cell stage and reappeared in fertilized embryos at the 8-cell stage and in NT and PA embryos at the 4-cell stage. H4K20me3 intensity in 4-cell, 8-cell, and morula stages of fertilized embryos was significantly lower than in NT and PA embryos, suggesting aberrant regulation of H4K20me3 in PA and NT embryos. Indeed, RNA expression of the H4K20 methyltransferase Suv4-20h2 in 4-cell fertilized embryos was significantly lower than NT embryos. Knockdown of Suv4-20h2 in NT embryos rescued the H4K20me3 pattern similar to fertilized embryos. Compared to control NT embryos, knockdown of Suv4-20h2 in NT embryos improved blastocyst development ratios (11.1% vs. 30.5%) and full-term cloning efficiencies (0.8% vs. 5.9%). Upregulation of reprogramming factors, including Kdm4b, Kdm4d, Kdm6a, and Kdm6b, as well as ZGA-related factors, including Dux, Zscan4, and Hmgpi, was observed with Suv4-20h2 knockdown in NT embryos. Collectively, these are the first findings to demonstrate that H4K20me3 is an epigenetic barrier of NT reprogramming and begin to unravel the epigenetic mechanisms of H4K20 trimethylation in cell plasticity during natural reproduction and NT reprogramming in mice.

Fractal dimension of the cortical gray matter outweighs other brain MRI features as a predictor of transition to dementia in patients with mild cognitive impairment and leukoaraiosis.

Background: The relative contribution of changes in the cerebral white matter (WM) and cortical gray matter (GM) to the transition to dementia in patients with mild cognitive impairment (MCI) is not yet established. In this longitudinal study, we aimed to analyze MRI features that may predict the transition to dementia in patients with MCI and T2 hyperintensities in the cerebral WM, also known as leukoaraiosis. Methods: Sixty-four participants with MCI and moderate to severe leukoaraiosis underwent baseline MRI examinations and annual neuropsychological testing over a 2 year period. The diagnosis of dementia was based on established criteria. We evaluated demographic, neuropsychological, and several MRI features at baseline as predictors of the clinical transition. The MRI features included visually assessed MRI features, such as the number of lacunes, microbleeds, and dilated perivascular spaces, and quantitative MRI features, such as volumes of the cortical GM, hippocampus, T2 hyperintensities, and diffusion indices of the cerebral WM. Additionally, we examined advanced quantitative features such as the fractal dimension (FD) of cortical GM and WM, which represents an index of tissue structural complexity derived from 3D-T1 weighted images. To assess the prediction of transition to dementia, we employed an XGBoost-based machine learning system using SHapley Additive exPlanations (SHAP) values to provide explainability to the machine learning model. Results: After 2 years, 18 (28.1%) participants had transitioned from MCI to dementia. The area under the receiving operator characteristic curve was 0.69 (0.53, 0.85) [mean (90% confidence interval)]. The cortical GM-FD emerged as the top-ranking predictive feature of transition. Furthermore, aggregated quantitative neuroimaging features outperformed visually assessed MRI features in predicting conversion to dementia. Discussion: Our findings confirm the complementary roles of cortical GM and WM changes as underlying factors in the development of dementia in subjects with MCI and leukoaraiosis. FD appears to be a biomarker potentially more sensitive than other brain features.

Seasonal variations in the metabolomic profile of the ovarian follicle components in Italian Mediterranean Buffaloes.

The aim of this work was to evaluate the seasonal effect on the metabolomic profile of the ovarian follicle in Italian Mediterranean buffalo to unravel the causes of the reduced competence during the non-breeding season (NBS). Samples of follicular fluid, follicular cells, cumulus cells and oocytes were collected from abattoir-derived ovaries during breeding season (BS) and NBS and analyzed by 1H Nuclear Magnetic Resonance. The Orthogonal Projections to Latent Structures of the Discriminant Analysis showed clear separation into seasonal classes and Variable Importance in Projection method identified differentially abundant metabolites between seasons. Seasonal differences were recorded in metabolite content in all analyzed components suggesting that the decreased oocyte competence during NBS may be linked to alteration of several metabolic pathways. The pathway enrichment analysis revealed that differences in the metabolites between the seasons were linked to glutathione, energy generating and amino acid metabolism and phospholipid biosynthesis. The current work allows the identification of potential positive competence markers in the follicular fluid as glutathione, glutamate, lactate and choline, and negative markers like leucine, isoleucine and ß-hydroxybutyrate. These results form a major basis to develop potential strategies to optimize the follicular environment and the IVM medium to improve the competence of oocytes during the NBS.

Epigenetic dynamics of H4K20me3 modification during oocyte maturation and early reprogramming of somatic cell nuclear transfer goat embryos.

OBJECTIVE: We examined the epigenetic dynamics of histone H4K20 trimethylation (H4K20me3), a repressive signature in heterochromatin, during goat oocyte meiosis and the reprogramming of somatic cell nuclear transfer (NT) embryos through the first three cell divisions. METHODS: Following NT, oocytes were treated with parthenogenetic activation (PA), by 5 µM calcium ionophore A23187 for 5 min followed by incubation in 2.0 mM 6-dimethylaminopurine with 5 µg/mL cycloheximide for 4 h. NT embryos up to 8-celled stage were incubated with H4K20me3 antibody. RESULTS: Immunofluorescence microscopy revealed the existence of a persistent H4K20me3 signature during oocyte maturation from germinal vesicle phase to metaphase I, anaphase I, telophase I, and metaphase II, with a gradual reduction in staining intensity. NT embryos at the 2-, 4- and 8-celled stage showed lower H4K20me3 intensity than PA and IVF embryos (P < 0.05). CONCLUSION: These results indicate that NT embryos exhibit insufficient H4K20me3 modification compared with IVF and PA embryos during early reprogramming, suggesting the existence of a resistant memory of differentiated cell nuclear architecture. These findings help unravel the epigenetic mechanism of histone H4K20me3 in goat nuclear transfer reprogramming.

Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos.

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

Paramagnetic rims are a promising diagnostic imaging biomarker in multiple sclerosis.

BACKGROUND: White matter lesions (WMLs) on brain magnetic resonance imaging (MRI) in multiple sclerosis (MS) may contribute to misdiagnosis. In chronic active lesions, peripheral iron-laden macrophages appear as paramagnetic rim lesions (PRLs). OBJECTIVE: To evaluate the sensitivity and specificity of PRLs in differentiating MS from mimics using clinical 3T MRI scanners. METHOD: This retrospective international study reviewed MRI scans of patients with MS (n = 254), MS mimics (n = 91) and older healthy controls (n = 217). WMLs, detected using fluid-attenuated inversion recovery MRI, were analysed with phase-sensitive imaging. Sensitivity and specificity were assessed for PRLs. RESULTS: At least one PRL was found in 22.9% of MS and 26.1% of clinically isolated syndrome (CIS) patients. Only one PRL was found elsewhere. The identification of ⩾1 PRL was the optimal cut-off and had high specificity (99.7%, confidence interval (CI) = 98.20%-99.99%) when distinguishing MS and CIS from mimics and healthy controls, but lower sensitivity (24.0%, CI = 18.9%-36.6%). All patients with a PRL showing a central vein sign (CVS) in the same lesion (n = 54) had MS or CIS, giving a specificity of 100% (CI = 98.8%-100.0%) but equally low sensitivity (21.3%, CI = 16.4%-26.81%). CONCLUSION: PRLs may reduce diagnostic uncertainty in MS by being a highly specific imaging diagnostic biomarker, especially when used in conjunction with the CVS.

Seasonal Variations in the Lipid Profile of the Ovarian Follicle in Italian Mediterranean Buffaloes.

The reduced oocyte competence recorded during the non-breading season (NBS) is one of the key factors affecting the profitability of buffalo farming and limits the IVEP efficiency. The purpose of this experiment was to evaluate whether season influences the lipid content within the ovarian follicle in the Italian Mediterranean buffalo. Abattoir-derived ovaries were collected during the breeding season (BS) and the NBS, and different matrices (follicular fluid, oocytes, cumulus and follicular cells) were recovered. After the extraction of the apolar fraction, all samples were analyzed by H1 nuclear magnetic resonance and FF samples by gas chromatography-mass spectrometry. Seasonal differences in lipid composition were observed in all matrices. In particular, during the NBS, the triglyceride content was higher in the follicular fluid and in the oocytes but reduced in the follicular cells. Both cholesterol and phospholipids were reduced in the follicular fluid and follicular cells during the NBS. Furthermore, the total amount of non-esterified fatty acids was significantly increased in the follicular fluid. The seasonal variation in lipid profile of the follicle may, in part, account for the reduced buffalo oocyte competence during the NBS, due to the critical role played by lipids in regulating ovarian functions.

Mild gray matter atrophy in patients with long-standing multiple sclerosis and favorable clinical course.

The mechanisms responsible for the favorable clinical course in multiple sclerosis (MS) remain unclear. In this longitudinal study, we assessed whether magnetic resonance imaging (MRI)-based changes in focal and diffuse brain damage are associated with a long-term favorable MS diseases course. We found that global brain and gray matter (GM) atrophy changes were milder in MS patients with long-standing disease (⩾30 years from onset) and favorable (no/minimal disability) clinical course than in sex-age-matched disable MS patients, independently of lesions accumulation. Data showed that different trajectories of volume changes, as reflected by mild GM atrophy, may characterize patients with long-term favorable evolution.

Effect of BDNF Val66Met polymorphism on hippocampal subfields in multiple sclerosis patients.

Brain-derived neurotrophic factor (BDNF) Val66Met polymorphism was shown to strongly affect BDNF function, but its role in modulating gray matter damage in multiple sclerosis (MS) patients is still not clear. Given BDNF relevance on the hippocampus, we aimed to explore BDNF Val66Met polymorphism effect on hippocampal subfield volumes and its role in cognitive functioning in MS patients. Using a 3T scanner, we obtained dual-echo and 3DT1-weighted sequences from 50 MS patients and 15 healthy controls (HC) consecutively enrolled. MS patients also underwent genotype analysis of BDNF, neurological and neuropsychological evaluation. Hippocampal subfields were segmented by using Freesurfer. The BDNF Val66Met polymorphism was found in 22 MS patients (44%). Compared to HC, MS patients had lower volume in: bilateral hippocampus-amygdala transition area (HATA); cornus ammonis (CA)1, granule cell layer of dentate gyrus (GCL-DG), CA4 and CA3 of the left hippocampal head; molecular layer (ML) of the left hippocampal body; presubiculum of right hippocampal body and right fimbria. Compared to BDNF Val66Val, Val66Met MS patients had higher volume in bilateral hippocampal tail; CA1, ML, CA3, CA4, and GCL-DG of left hippocampal head; CA1, ML, and CA3 of the left hippocampal body; left HATA and presubiculum of the right hippocampal head. In MS patients, higher lesion burden was associated with lower volume of presubiculum of right hippocampal body; lower volume of left hippocampal tail was associated with worse visuospatial memory performance; lower volume of left hippocampal head with worse performance in semantic fluency. Our findings suggest the BNDF Val66Met polymorphism may have a protective role in MS patients against both hippocampal atrophy and cognitive impairment. BDNF genotype might be a potential biomarker for predicting cognitive prognosis, and an interesting target to study for neuroprotective strategies.

MRI Prognostic Factors in Multiple Sclerosis, Neuromyelitis Optica Spectrum Disorder, and Myelin Oligodendrocyte Antibody Disease.

Several MRI measures have been developed in the last couple of decades, providing a number of imaging biomarkers that can capture the complexity of the pathological processes occurring in multiple sclerosis (MS) brains. Such measures have provided more specific information on the heterogeneous pathologic substrate of MS-related tissue damage, being able to detect, and quantify the evolution of structural changes both within and outside focal lesions. In clinical practise, MRI is increasingly used in the MS field to help to assess patients during follow-up, guide treatment decisions and, importantly, predict the disease course. Moreover, the process of identifying new effective therapies for MS patients has been supported by the use of serial MRI examinations in order to sensitively detect the sub-clinical effects of disease-modifying treatments at an earlier stage than is possible using measures based on clinical disease activity. However, despite this has been largely demonstrated in the relapsing forms of MS, a poor understanding of the underlying pathologic mechanisms leading to either progression or tissue repair in MS as well as the lack of sensitive outcome measures for the progressive phases of the disease and repair therapies makes the development of effective treatments a big challenge. Finally, the role of MRI biomarkers in the monitoring of disease activity and the assessment of treatment response in other inflammatory demyelinating diseases of the central nervous system, such as neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte antibody disease (MOGAD) is still marginal, and advanced MRI studies have shown conflicting results. Against this background, this review focused on recently developed MRI measures, which were sensitive to pathological changes, and that could best contribute in the future to provide prognostic information and monitor patients with MS and other inflammatory demyelinating diseases, in particular, NMOSD and MOGAD.

Efficient mutagenesis targeting the IFNAR1 gene in mice using a combination of Cas9 protein and dual gRNAs.

We injected mouse zygotes with combinations of Cas9 protein, Cas9 mRNA, and two gRNAs targeting a single exon of type I interferon receptor (IFNAR1) to determine the gene targeting efficiencies. Cas9 protein produced on-target mutations more efficiently than Cas9 mRNA when each was used with a single gRNA, regardless of which gRNA was used. When Cas9 mRNA and Cas9 protein were co-injected, the on-target efficiency could reach 97.0% when both gRNAs were used, which was higher than when either gRNA was used alone (61.3% and 75.5%, respectively; P<0.05). Co-injection of Cas9 protein with both gRNAs produced the highest on-target mutation rate of any combination (100.0%). Most on-target mutations were deletions of 2 to 113 nucleotides, and there were few off-target mutations in mutant animals. The expression intensity of IFNAR1 was reduced in heterozygous IFNAR1 +/- mice (IF) and almost or completely absent in homozygous null IFNAR -/- mice compared with that in wild-type mice (IF and Western blot). When both gRNAs targeting IFNAR1 were used simultaneously with two gRNAs targeting FVII, the on-target editing efficiency on each gene was 96.8% and 85.5%, respectively. Co-injection of dual gRNAs and Cas9 protein is an efficient approach for IFNAR1 knockout and multi-gene editing in mice and may be applied in other animal models and breeding livestock.

Intracranial Pressure Monitoring in Poor-Grade Patients with Aneurysmal Subarachnoid Hemorrhage Treated by Coiling.

OBJECTIVE: The main objective of the present study was to analyze the intracranial pressure (ICP) and cerebral perfusion pressure (CPP) changes during coiling. We also evaluated the prevalence of rebleeding and outcomes for patients monitored before and after coiling. METHODS: Ninety-nine consecutive poor-grade patients with aneurysmal subarachnoid hemorrhage (aSAH; World Federation of Neurological Surgeons grade IV and V) were enrolled in our prospective observational study. For 31 patients, ICP and CPP monitoring was started immediately after the diagnosis of aSAH, and the values were recorded every 15 minutes during coiling (early ICP group). For 68 patients, ICP and CPP monitoring began after coiling (late ICP group). The outcomes were evaluated at 90 days using the modified Rankin scale. RESULTS: At the beginning of coiling, the ICP was >20 mm Hg in 10 patients (35.7%). The median ICP was 18 mm Hg (range, 5-60 mm Hg). The CPP was <60 mm Hg in 6 patients (24%). The median CPP was 70 mm Hg (range, 30-101 mm Hg). Despite medical treatment and/or cerebrospinal fluid drainage, 51.6% of the patients monitored during coiling had at least one episode of intracranial hypertension (defined as ICP >20 mm Hg), and 51.6% had at least one episode of reduced CPP (defined as CPP <60 mm Hg). Early monitoring (before aneurysm repair) was not associated with rebleeding. At 90 days, the functional recovery was better in the early ICP group (P = 0.004). CONCLUSIONS: During coiling, patients with poor-grade aSAH can experience episodes of intracranial hypertension and reduced CPP. Early and appropriate treatment of elevated ICP was not associated with rebleeding and might have improved the outcomes.

Site specificity of blastocyst hatching significantly influences pregnancy outcomes in mice.

Blastocysts hatch from the zona pellucida (ZP) to enable implantation into the uterine endometrial epithelium, but little is known regarding the effect of hatching sites on pregnancy outcomes. Murine hatching embryos were categorized into five groups based on initial trophectoderm projection (TEP)/ZP position corresponding to the inner cell mass center. In blastocysts (3.5 dpc) post-12 hours in vitro culture, TEP rates of A-site (44.4%) and B-site (38.6%) embryos were higher than those of C-site (12.5%) and D-site (3.1%) embryos, while the O-site (1.4%) was the lowest (P < .05). Post-ET A-site (55.6%) and B-site (65.6%) birth rates were higher than those of C-site embryos (21.3%) and controls (P < .05). Furthermore, live birth rate of B-site embryos remained higher than C-site embryos (68.8% vs 31.3%; P < .05) when both were transferred into the same recipients. Different TEP site blastocysts exhibited different implantation competences: the implantation rate of C-site embryos was lower than that of both A- and B-site groups (67.7% vs 84.3% and 83.2%, respectively; P < .05) at 2 days post-ET. C-site embryos also had a distinctly higher ratio of developmental defects (47.5%) than A- and B-site embryos (22.5% and 14.6%, respectively), with implantation failure mainly associated with poor birth rate, a finding corroborated by differential gene expression analysis such as LIF, LIFR, and S100a9. Surprisingly, acidified Tyrode's solution (AAH)-treated B-site blastocysts had a significantly increased birth rate (77.1%) than C-site (55.3%) and controls (43.4%). Site specificity and differential gene expression during embryo hatching can be applied in ART screening. More importantly, assisted hatching by AAH is effective and feasible for improving pregnancy and term development, particularly at the B-site, for humans and in animal husbandry.

Structural and Functional Connectivity Substrates of Cognitive Impairment in Multiple Sclerosis.

Cognitive impairment (CI) occurs in 43 to 70% of multiple sclerosis (MS) patients at both early and later disease stages. Cognitive domains typically involved in MS include attention, information processing speed, memory, and executive control. The growing use of advanced magnetic resonance imaging (MRI) techniques is furthering our understanding on the altered structural connectivity (SC) and functional connectivity (FC) substrates of CI in MS. Regarding SC, different diffusion tensor imaging (DTI) measures (e.g., fractional anisotropy, diffusivities) along tractography-derived white matter (WM) tracts showed relevance toward CI. Novel diffusion MRI techniques, including diffusion kurtosis imaging, diffusion spectrum imaging, high angular resolution diffusion imaging, and neurite orientation dispersion and density imaging, showed more pathological specificity compared to the traditional DTI but require longer scan time and mathematical complexities for their interpretation. As for FC, task-based functional MRI (fMRI) has been traditionally used in MS to brain mapping the neural activity during various cognitive tasks. Analysis methods of resting fMRI (seed-based, independent component analysis, graph analysis) have been applied to uncover the functional substrates of CI in MS by revealing adaptive or maladaptive mechanisms of functional reorganization. The relevance for CI in MS of SC-FC relationships, reflecting common pathogenic mechanisms in WM and gray matter, has been recently explored by novel MRI analysis methods. This review summarizes recent advances on MRI techniques of SC and FC and their potential to provide a deeper understanding of the pathological substrates of CI in MS.

Principles of Sustainable Healthy Diets in Worldwide Dietary Guidelines: Efforts So Far and Future Perspectives.

Food choices and eating behaviours have a large impact on both human and planetary health. Recently, the Food and Agricultural Organisation (FAO) of the United Nations and the World Health Organisation have developed a list of 16 guiding principles to achieve sustainable healthy diets (SHDs). They proposed that development of food-based dietary guidelines (FBDGs) should be a core element in the implementation of these SHDs in each country. The objective of this review is to explore the degree of alignment of current FBDGs to these guiding principles. A total of 43 FBDGs, written or translated into English, were collected from the online repository developed by the FAO and were analysed for their adherence to each of the guiding principles. Results were stratified for period of publication and geographical macro-area. Overall, there were high levels of inclusion of the factors related to health outcomes, especially in the most recent FBDGs. Conversely, environmental impact and socio-cultural aspects of diet were considered less frequently, especially in the older FBDGs. These results highlight the importance of revising FBDGs, especially to include emerging topics which represent the areas with the highest scope for improvement in the future versions of FBDGs. Replication of the present study in the coming years will be worthwhile to monitor improvements in the adherence of global FBDGs to the guiding principles of SHDs. The attainment of such a goal could promote a more rapid transition towards SHDs, as well as highlighting pivotal research trajectories to increase adoption and evaluate the impact on the food system.

Treatment of Hydatid Liver Cyst With Double Percutaneous Aspiration and Ethanol Injection Under Ultrasound Guidance: 6.5-Year Median Follow-up Analysis.

PURPOSE: Although hydatid liver cyst (HLC) is a benign disease, treatment is recommended to avoid life-threatening complications. There are several treatment options for HLC: "wait-and-watch," medical or surgical or percutaneous treatment. The purpose of this study was to assess the long-term effectiveness of an alternative of the traditional percutaneous PAIR procedure, called double percutaneous aspiration and ethanol injection (D-PAI). MATERIALS AND METHODS: This prospective, non-randomized study was conducted from 1988 to 2019 using DPAI procedure characterized by no reaspiration of the ethanol injected to replace the aspirated fluid and repetition of the procedure after 3-7 days. RESULTS: Two hundred and three patients with 290 HLCs underwent D-PAI. Two hundred and two HLC (160 patients) were univesicular cysts and 88 (43 patient) were multivesicular. Seventeen patients underwent one D-PAI session, 15 patients two sessions, and 18 up to four sessions. The follow-up ranged 0.9-21 years (median 6.5 years). On ultrasound, 188 cysts (64.8%) disappeared; 57 cysts (19.7%) became solid (inactive) and 45 (15.5%) showed a small inactive residual component. Parasitologic cure was very high. The overall response to D-PAI was higher than 90% considering also the procedures carried out after the first D-PAI at the time of recurrence. One patient died for anaphylactic shock. The hospital stay ranged 1-3 days. Smaller cysts (< 5 cm) healed sooner than larger cysts (p < 0.001). CONCLUSIONS: Long-term analysis showed that D-PAI is a safe and effective option in percutaneous treatment of viable HLC, except for CE2/CE3b in which the recurrences can be observed. This inexpensive and simple procedure can be applied everywhere and especially in developing countries.

Dynamics of pseudo-atrophy in RRMS reveals predominant gray matter compartmentalization.

OBJECTIVE: To assess the dynamics of "pseudo-atrophy," the accelerated brain volume loss observed after initiation of anti-inflammatory therapies, in patients with multiple sclerosis (MS). METHODS: Monthly magnetic resonance imaging (MRI) data of patients from the IMPROVE clinical study (NCT00441103) comparing relapsing-remitting MS patients treated with interferon beta-1a (IFNß-1a) for 40 weeks versus those receiving placebo (16 weeks) and then IFNß-1a (24 weeks) were used to assess percentage of gray (PGMVC) and white matter (PWMVC) volume changes. Comparisons of PGMVC and PWMVC slopes were performed with a mixed effect linear model. In the IFNß-1a-treated arm, a quadratic term was included in the model to evaluate the plateauing effect over 40 weeks. RESULTS: Up to week 16, PGMVC was -0.14% per month in the placebo and -0.27% per month in treated patients (P < 0.001). Over the same period, the decrease in PWMVC was -0.067% per month in the placebo and -0.116% per month in treated patients (P = 0.27). Similar changes were found in the group originally randomized to placebo when starting IFNß-1a treatment (week 16-40, reliability analysis). In the originally treated group, over 40 weeks, the decrease in PGMVC showed a significant (P < 0.001) quadratic component, indicating a plateauing at week 20. INTERPRETATION: Findings reported here add new insights into the complex mechanisms of pseudo-atrophy and its relation to the compartmentalized inflammation occurring in the GM of MS patients. Ongoing and forthcoming clinical trials including MRI-derived GM volume loss as an outcome measure need to account for potentially significant GM volume changes as part of the initial treatment effect.

Identifying the Distinct Cognitive Phenotypes in Multiple Sclerosis.

Importance: Cognitive impairment is a common and disabling feature of multiple sclerosis (MS), but a precise characterization of cognitive phenotypes in patients with MS is lacking. Objectives: To identify cognitive phenotypes in a clinical cohort of patients with MS and to characterize their clinical and magnetic resonance imaging (MRI) features. Design, Setting, and Participants: This multicenter cross-sectional study consecutively screened clinically stable patients with MS and healthy control individuals at 8 MS centers in Italy from January 1, 2010, to October 31, 2019. Patients with MS and healthy control individuals who were not using psychoactive drugs and had no history of other neurological or medical disorders, learning disability, severe head trauma, and alcohol or drug abuse were enrolled. Main Outcomes and Measures: Participants underwent a neurological examination and a cognitive evaluation with the Rao Brief Repeatable Battery and Stroop Color and Word Test. A subgroup of participants also underwent a brain MRI examination. Latent profile analysis was used on cognitive test z scores to identify cognitive phenotypes. Linear regression and mixed-effects models were used to define clinical and MRI features of each phenotype. Results: A total of 1212 patients with MS (mean [SD] age, 41.1 [11.1] years; 784 women [64.7%]) and 196 healthy control individuals (mean [SD] age, 40.4 [8.6] years; 130 women [66.3%]) were analyzed in this study. Five cognitive phenotypes were identified: preserved cognition (n = 235 patients [19.4%]), mild-verbal memory/semantic fluency (n = 362 patients [29.9%]), mild-multidomain (n = 236 patients [19.5%]), severe-executive/attention (n = 167 patients [13.8%]), and severe-multidomain (n = 212 patients [17.5%]) involvement. Patients with preserved cognition and mild-verbal memory/semantic fluency were younger (mean [SD] age, 36.5 [9.8] years and 38.2 [11.1] years) and had shorter disease duration (mean [SD] 8.0 [7.3] years and 8.3 [7.6] years) compared with patients with mild-multidomain (mean [SD] age, 42.6 [11.2] years; mean [SD] disease duration, 12.8 [9.6] years; P < .001), severe-executive/attention (mean [SD] age, 42.9 [11.7] years; mean [SD] disease duration, 12.2 [9.5] years; P < .001), and severe-multidomain (mean [SD] age, 44.0 [11.0] years; mean [SD] disease duration, 13.3 [10.2] years; P < .001) phenotypes. Severe cognitive phenotypes prevailed in patients with progressive MS. At MRI evaluation, compared with those with preserved cognition, patients with mild-verbal memory/semantic fluency exhibited decreased mean (SE) hippocampal volume (5.42 [0.68] mL vs 5.13 [0.68] mL; P = .04), patients with the mild-multidomain phenotype had decreased mean (SE) cortical gray matter volume (687.69 [35.40] mL vs 662.59 [35.48] mL; P = .02), patients with severe-executive/attention had higher mean (SE) T2-hyperintense lesion volume (51.33 [31.15] mL vs 99.69 [34.07] mL; P = .04), and patients with the severe-multidomain phenotype had extensive brain damage, with decreased volume in all the brain structures explored, except for nucleus pallidus, amygdala and caudate nucleus. Conclusions and Relevance: This study found that by defining homogeneous and clinically meaningful phenotypes, the limitations of the traditional dichotomous classification in MS can be overcome. These phenotypes can represent a more meaningful measure of the cognitive status of patients with MS and can help define clinical disability, support clinicians in treatment choices, and tailor cognitive rehabilitation strategies.