Normal-appearing Cortical Gray Matter Research Articles

Identification of Y‒linked biomarkers and exploration of immune infiltration of normal-appearing gray matter in multiple sclerosis by bioinformatic analysis

BackgroundThe knowledge of normal‒appearing cortical gray matter (NAGM) in multiple sclerosis (MS) remains unclear. In this study, we aimed to identify diagnostic biomarkers and explore the immune infiltration characteristics of NAGM in MS through bioinformatic analysis and validation in vivo. MethodsDifferentially expressed genes (DEGs) were analyzed. Subsequently, the functional pathways of the DEGs were determined. After screening the overlapping DEGs of MS with two machine learning methods, the biomarkers’ efficacy and the expression levels of overlapping DEGs were calculated. Quantitative reverse transcription polymerase chain reaction (qRT‒PCR) identified the robust diagnostic biomarkers. Additionally, infiltrating immune cell populations were estimated and correlated with the biomarkers. Finally, the characteristics of immune infiltration of NAGM from MS were evaluated. ResultsA total of 98 DEGs were identified. They participated in sensory transduction of the olfactory system, synaptic signaling, and immune responses. Nine overlapping genes were screened by machine learning methods. After verified by ROC curve, four genes, namely HLA‒DRB1, RPS4Y1, EIF1AY and USP9Y, were screened as candidate biomarkers. The mRNA expression of RPS4Y1 and USP9Y was significantly lower in MS patients than that in the controls. They were selected as the robust diagnostic biomarkers for male MS patients. RPS4Y1 and USP9Y were both positively correlated with memory B cells. Moreover, naive CD4+ T cells and monocytes were increased in the NAGM of MS patients compared with those in controls. ConclusionsLow expressed Y‒linked genes, RPS4Y1 and USP9Y, were identified as diagnostic biomarkers for MS in male patients. The inhomogeneity of immune cells in NAGM might exacerbate intricate interplay between the CNS and the immune system in the MS.

Personalized maps of T1 relaxometry abnormalities provide correlates of disability in multiple sclerosis patients

Objectives and aimsQuantitative MRI (qMRI) has greatly improved the sensitivity and specificity of microstructural brain pathology in multiple sclerosis (MS) when compared to conventional MRI (cMRI). More than cMRI, qMRI also provides means to assess pathology within the normal-appearing and lesion tissue. In this work, we further developed a method providing personalized quantitative T1 (qT1) abnormality maps in individual MS patients by modeling the age dependence of qT1 alterations. In addition, we assessed the relationship between qT1 abnormality maps and patients’ disability, in order to evaluate the potential value of this measurement in clinical practice. MethodsWe included 119 MS patients (64 relapsing-remitting MS (RRMS), 34 secondary progressive MS (SPMS), 21 primary progressive MS (PPMS)), and 98 Healthy Controls (HC). All individuals underwent 3T MRI examinations, including Magnetization Prepared 2 Rapid Acquisition Gradient Echoes (MP2RAGE) for qT1 maps and High-Resolution 3D Fluid Attenuated Inversion Recovery (FLAIR) imaging. To calculate personalized qT1 abnormality maps, we compared qT1 in each brain voxel in MS patients to the average qT1 obtained in the same tissue (grey/white matter) and region of interest (ROI) in healthy controls, hereby providing individual voxel-based Z-score maps. The age dependence of qT1 in HC was modeled using linear polynomial regression. We computed the average qT1 Z-scores in white matter lesions (WMLs), normal-appearing white matter (NAWM), cortical grey matter lesions (GMcLs) and normal-appearing cortical grey matter (NAcGM). Lastly, a multiple linear regression (MLR) model with the backward selection including age, sex, disease duration, phenotype, lesion number, lesion volume and average Z-score (NAWM/NAcGM/WMLs/GMcLs) was used to assess the relationship between qT1 measures and clinical disability (evaluated with EDSS). ResultsThe average qT1 Z-score was higher in WMLs than in NAWM. (WMLs: 1.366 ± 0.409, NAWM: −0.133 ± 0.288, [mean ± SD], p < 0.001). The average Z-score in NAWM in RRMS patients was significantly lower than in PPMS patients (p = 0.010). The MLR model showed a strong association between average qT1 Z-scores in white matter lesions (WMLs) and EDSS (R2 = 0.549, β = 0.178, 97.5 % CI = 0.030 to 0.326, p = 0.019). Specifically, we measured a 26.9 % increase in EDSS per unit of qT1 Z-score in WMLs in RRMS patients (R2 = 0.099, β = 0.269, 97.5 % CI = 0.078 to 0.461, p = 0.007). ConclusionsWe showed that personalized qT1 abnormality maps in MS patients provide measures related to clinical disability, supporting the use of those maps in clinical practice.

Evaluating brain damage in multiple sclerosis with simultaneous multi-angular-relaxometry of tissue.

ObjectiveMultiple sclerosis (MS) is a common demyelinating central nervous system disease. MRI methods that can quantify myelin loss are needed for trials of putative remyelinating agents. Quantitative magnetization transfer MRI introduced the macromolecule proton fraction (MPF), which correlates with myelin concentration. We developed an alternative approach, Simultaneous‐Multi‐Angular‐Relaxometry‐of‐Tissue (SMART) MRI, to generate MPF. Our objective was to test SMART‐derived MPF metric as a potential imaging biomarker of demyelination.MethodsTwenty healthy control (HC), 11 relapsing–remitting MS (RRMS), 22 progressive MS (PMS), and one subject with a biopsied tumefactive demyelinating lesion were scanned at 3T using SMART MRI. SMART‐derived MPF metric was determined in normal‐appearing cortical gray matter (NAGM), normal‐appearing subcortical white matter (NAWM), and demyelinating lesions. MPF metric was evaluated for correlations with physical and cognitive test scores. Comparisons were made between HC and MS and between MS subtypes. Furthermore, correlations were determined between MPF and neuropathology in the biopsied person.ResultsSMART‐derived MPF in NAGM and NAWM were lower in MS than HC (p < 0.001). MPF in NAGM, NAWM and lesions differentiated RRMS from PMS (p < 0.01, p < 0.001, p < 0.001, respectively), whereas lesion volumes did not. MPF in NAGM, NAWM and lesions correlated with the Expanded Disability Status Scale (p < 0.01, p < 0.001, p < 0.001, respectively) and nine‐hole peg test (p < 0.001, p < 0.001, p < 0.01, respectively). MPF was lower in the histopathologically confirmed inflammatory demyelinating lesion than the contralateral NAWM and increased in the biopsied lesion over time, mirroring improved clinical performance.InterpretationSMART‐derived MPF metric holds potential as a quantitative imaging biomarker of demyelination and remyelination.

Increased HLA-DR expression and cortical demyelination in MS links with HLA-DR15.

ObjectiveTo investigate molecular changes in multiple sclerosis (MS) normal-appearing cortical gray matter (NAGM).MethodsWe performed a whole-genome gene expression microarray analysis of human brain autopsy tissues from 64 MS NAGM samples and 42 control gray matter samples. We further examined our cases by HLA genotyping and performed immunohistochemical and immunofluorescent analysis of all human brain tissues.ResultsHLA-DRB1 is the transcript with highest expression in MS NAGM with a bimodal distribution among the examined cases. Genotyping revealed that every case with the MS-associated HLA-DR15 haplotype also shows high HLA-DRB1 expression and also of the tightly linked HLA-DRB5 allele. Quantitative immunohistochemical analysis confirmed the higher expression of HLA-DRB1 in HLA-DRB1*15:01 cases at the protein level. Analysis of gray matter lesion size revealed a significant increase of cortical lesion size in cases with high HLA-DRB1 expression.ConclusionsOur data indicate that increased HLA-DRB1 and -DRB5 expression in the brain of patients with MS may be an important factor in how the HLA-DR15 haplotype contributes to MS pathomechanisms in the target organ.

Multiparameter MRI quantification of microstructural tissue alterations in multiple sclerosis.

ObjectivesConventional MRI is not sensitive to many pathological processes underpinning multiple sclerosis (MS) ongoing in normal appearing brain tissue (NABT). Quantitative MRI (qMRI) and a multiparameter mapping (MPM) protocol are used to simultaneously quantify magnetization transfer (MT) saturation, transverse relaxation rate R2* (1/T2*) and longitudinal relaxation rate R1 (1/T1), and assess differences in NABT microstructure between MS patients and healthy controls (HC).MethodsThis prospective cross-sectional study involves 36 MS patients (21 females, 15 males; age range 22–63 years; 15 relapsing-remitting MS - RRMS; 21 primary or secondary progressive MS - PMS) and 36 age-matched HC (20 females, 16 males); age range 21–61 years). The qMRI maps are computed and segmented in lesions and 3 normal appearing cerebral tissue classes: normal appearing cortical grey matter (NACGM), normal appearing deep grey matter (NADGM), normal appearing white matter (NAWM). Individual median values are extracted for each tissue class and MR parameter. MANOVAs and stepwise regressions assess differences between patients and HC.ResultsMS patients are characterized by a decrease in MT, R2* and R1 within NACGM (p < .0001) and NAWM (p < .0001). In NADGM, MT decreases (p < .0001) but R2* and R1 remain normal. These observations tend to be more pronounced in PMS. Quantitative MRI parameters are independent predictors of clinical status: EDSS is significantly related to R1 in NACGM and R2* in NADGM; the latter also predicts motor score. Cognitive score is best predicted by MT parameter within lesions.ConclusionsMultiparametric data of brain microstructure concord with the literature, predict clinical performance and suggest a diffuse reduction in myelin and/or iron content within NABT of MS patients.

Beyond focal cortical lesions in MS: An in vivo quantitative and spatial imaging study at 7T.

Using quantitative T2* 7-tesla (7T) MRI as a marker of demyelination and iron loss, we investigated, in patients with relapsing-remitting multiple sclerosis (RRMS) and secondary progressive multiple sclerosis (SPMS), spatial and tissue intrinsic characteristics of cortical lesion(s) (CL) types, and structural integrity of perilesional normal-appearing cortical gray matter (NACGM) as a function of distance from lesions. Patients with MS (18 RRMS, 11 SPMS), showing at least 2 CL, underwent 7T T2* imaging to obtain (1) magnitude images for segmenting focal intracortical lesion(s) (ICL) and leukocortical lesion(s) (LCL), and (2) cortical T2* maps. Anatomical scans were collected at 3T for cortical surface reconstruction using FreeSurfer. Seventeen age-matched healthy participants served as controls. ICL were predominantly located in sulci of frontal, parietal, and cingulate cortex; LCL distribution was more random. In MS, T2* was higher in both ICL and LCL, indicating myelin and iron loss, than in NACGM (p < 0.00003) irrespective of CL subtype and MS phenotype. T2* was increased in perilesional cortex, tapering away from CL toward NACGM, the wider changes being for LCL in SPMS. NACGM T2* was higher in SPMS relative to RRMS (p = 0.006) and healthy cortex (p = 0.02). CL had the same degree of demyelination and iron loss regardless of lesion subtype and disease stage. Cortical damage expanded beyond visible CL, close to lesions in RRMS, and more diffusely in SPMS. Evaluation of NACGM integrity, beyond focal CL, could represent a surrogate marker of MS progression.

Metabolic gene expression changes in astrocytes in Multiple Sclerosis cerebral cortex are indicative of immune-mediated signaling.

Emerging as an important correlate of neurological dysfunction in Multiple Sclerosis (MS), extended focal and diffuse gray matter abnormalities have been found and linked to clinical manifestations such as seizures, fatigue and cognitive dysfunction. To investigate possible underlying mechanisms we analyzed the molecular alterations in histopathological normal appearing cortical gray matter (NAGM) in MS. By performing a differential gene expression analysis of NAGM of control and MS cases we identified reduced transcription of astrocyte specific genes involved in the astrocyte-neuron lactate shuttle (ANLS) and the glutamate-glutamine cycle (GGC). Additional quantitative immunohistochemical analysis demonstrating a CX43 loss in MS NAGM confirmed a crucial involvement of astrocytes and emphasizes their importance in MS pathogenesis. Concurrently, a Toll-like/IL-1β signaling expression signature was detected in MS NAGM, indicating that immune-related signaling might be responsible for the downregulation of ANLS and GGC gene expression in MS NAGM. Indeed, challenging astrocytes with immune stimuli such as IL-1β and LPS reduced their ANLS and GGC gene expression in vitro. The detected upregulation of IL1B in MS NAGM suggests inflammasome priming. For this reason, astrocyte cultures were treated with ATP and ATP/LPS as for inflammasome activation. This treatment led to a reduction of ANLS and GGC gene expression in a comparable manner. To investigate potential sources for ANLS and GGC downregulation in MS NAGM, we first performed an adjuvant-driven stimulation of the peripheral immune system in C57Bl/6 mice in vivo. This led to similar gene expression changes in spinal cord demonstrating that peripheral immune signals might be one source for astrocytic gene expression changes in the brain. IL1B upregulation in MS NAGM itself points to a possible endogenous signaling process leading to ANLS and GGC downregulation. This is supported by our findings that, among others, MS NAGM astrocytes express inflammasome components and that astrocytes are capable to release Il-1β in-vitro. Altogether, our data suggests that immune signaling of immune- and/or central nervous system origin drives alterations in astrocytic ANLS and GGC gene regulation in the MS NAGM. Such a mechanism might underlie cortical brain dysfunctions frequently encountered in MS patients.

A gradient in cortical pathology in multiple sclerosis by in vivo quantitative 7 T imaging.

We used a surface-based analysis of T2* relaxation rates at 7 T magnetic resonance imaging, which allows sampling quantitative T2* throughout the cortical width, to map in vivo the spatial distribution of intracortical pathology in multiple sclerosis. Ultra-high resolution quantitative T2* maps were obtained in 10 subjects with clinically isolated syndrome/early multiple sclerosis (≤ 3 years disease duration), 18 subjects with relapsing-remitting multiple sclerosis (≥ 4 years disease duration), 13 subjects with secondary progressive multiple sclerosis, and in 17 age-matched healthy controls. Quantitative T2* maps were registered to anatomical cortical surfaces for sampling T2* at 25%, 50% and 75% depth from the pial surface. Differences in laminar quantitative T2* between each patient group and controls were assessed using general linear model (P < 0.05 corrected for multiple comparisons). In all 41 multiple sclerosis cases, we tested for associations between laminar quantitative T2*, neurological disability, Multiple Sclerosis Severity Score, cortical thickness, and white matter lesions. In patients, we measured, T2* in intracortical lesions and in the intracortical portion of leukocortical lesions visually detected on 7 T scans. Cortical lesional T2* was compared with patients' normal-appearing cortical grey matter T2* (paired t-test) and with mean cortical T2* in controls (linear regression using age as nuisance factor). Subjects with multiple sclerosis exhibited relative to controls, independent from cortical thickness, significantly increased T2*, consistent with cortical myelin and iron loss. In early disease, T2* changes were focal and mainly confined at 25% depth, and in cortical sulci. In later disease stages T2* changes involved deeper cortical laminae, multiple cortical areas and gyri. In patients, T2* in intracortical and leukocortical lesions was increased compared with normal-appearing cortical grey matter (P < 10(-10) and P < 10(-7)), and mean cortical T2* in controls (P < 10(-5) and P < 10(-6)). In secondary progressive multiple sclerosis, T2* in normal-appearing cortical grey matter was significantly increased relative to controls (P < 0.001). Laminar T2* changes may, thus, result from cortical pathology within and outside focal cortical lesions. Neurological disability and Multiple Sclerosis Severity Score correlated each with the degree of laminar quantitative T2* changes, independently from white matter lesions, the greatest association being at 25% depth, while they did not correlate with cortical thickness and volume. These findings demonstrate a gradient in the expression of cortical pathology throughout stages of multiple sclerosis, which was associated with worse disability and provides in vivo evidence for the existence of a cortical pathological process driven from the pial surface.

Arterial spin labeling of hemangioblastoma: differentiation from metastatic brain tumors based on quantitative blood flow measurement

Hemangioblastoma and metastatic tumors are the major differential diagnoses for the posterior fossa tumors in adults. Our purpose was to evaluate the efficacy of ASL in differentiating hemangioblastomas from metastatic brain tumors. A total of 19 patients including 5 with a hemangioblastomas and 14 with metastatic tumors (7 from lung cancer, 4 from breast cancer, 1 from RCC, 1 from gastric cancer, and 1 from unknown origin) were enrolled in this study. ASL was performed using a pulsed ASL method at a 3-T unit. aTBF was measured as a mean absolute blood flow value within a region of interest drawn in the tumor. In addition, rTBF was obtained by normalizing the aTBF by a blood flow measured in the normal-appearing cortical gray matter. The aTBF and rTBF values were compared between hemangioblastomas and metastatic tumors using Student's t test. Both the aTBF and rTBF values were significantly higher in hemangioblastomas (mean aTBF ± SD = 437 ± 274mL/100g/min, mean rTBF ± SD = 7.96 ± 3.12) in comparison with metastatic brain tumors (mean aTBF ± SD = 125 ± 134mL/100g/min, mean rTBF ± SD = 2.98 ± 3.91; p < 0.05, respectively). However, a metastasis from RCC showed very high aTBF (559mL/100g/min) and rTBF (16.2). Our results demonstrated that ASL provides useful information to differentiate between hemangioblastomas and metastatic brain tumors. Metastasis from RCC may mimic hemangioblastoma on ASL blood flow measurement.

Differential Changes in Deep and Cortical Gray Matters of Patients With Multiple Sclerosis

The objective of our study was to evaluate the changes in quantitative diffusion tensor (DT) metrics and normalized T2-signal intensity (nT2-SI) values of normal-appearing cortical gray matter (CGM) and deep gray matter (DGM) in patients with multiple sclerosis (MS). Fifty patients with MS and 25 patients with no MS matched on sex/age were selected as controls. Conventional magnetic resonance imaging and DT imaging were performed. Fractional anisotropy (FA)/mean diffusivity (MD) and nT2-SI values of CGM and DGM were measured. Analyses of variance between the 2 groups were analyzed; Pearson correlations between DT metrics and nT2-SI values and brain parenchymal fraction (BPF) and T2 lesion volumes (LVs) were used. Patients with MS showed larger MD/smaller FA values in the CGM region compared with controls (P < 0.05). However, MD/FA values were not statistically significant in the DGM between MS and healthy control group. In DGM of MS patients, a significant decrease of nT2-SI values were observed when compared with controls (P < 0.05), but nT2-SI values in the CGM of MS patients showed no significant decrease. In CGM, only MD values of frontal lobes in MS patients were significantly (negatively) correlated with BPF(right: P = 0.009, left: P = 0.036) or T2 LVs (right: P = 0.002, left: P = 0.047). Normalized T2-SI values in all DGM regions of MS patients were significantly correlated with BPF (r = 0.282-0.504, P < 0.05) except for the left thalamus and bilateral red nucleus. There was no correlation between nT2-SI in all DGM regions and T2 LVs of MS patients. In CGM, the change in quantitative DT metrics of MS patients and the association with BPF and T2 LVs suggest the existence of microstructural destruction corresponding to inflammation, demyelination, or wallerian degeneration, but the changes in CGM were independent of the concomitant changes in BPF and T2 lesion. In DGM, a decrease of nT2-SI in MS patients and the correlation of nT2-SI values with BPF (brain atrophy) suggest excessive iron deposition related to chronic destruction. Our investigation indicates the possibility of different mechanism of pathological change in CGM and DGM.