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Repetitive transcranial magnetic stimulation (rTMS) is emerging as a non-invasive therapeutic strategy in the battle against Alzheimer’s disease. Alzheimer’s disease patients primarily show alterations of the default mode network for which the precuneus is a key node. Here, we hypothesized that targeting the precuneus with TMS represents a promising strategy to slow down cognitive and functional decline in Alzheimer’s disease patients. We performed a randomized, double-blind, sham-controlled, phase 2, 24-week trial to determine the safety and efficacy of precuneus stimulation in patients with mild-to-moderate Alzheimer’s disease. Fifty Alzheimer’s disease patients were randomly assigned in a 1:1 ratio to either receive precuneus or sham rTMS (mean age 73.7 years; 52% female). The trial included a 24-week treatment, with a 2-week intensive course in which rTMS (or sham) was applied daily five times per week, followed by a 22-week maintenance phase in which stimulation was applied once weekly. The Clinical Dementia Rating Scale–Sum of Boxes was selected as the primary outcome measure, in which post-treatment scores were compared to baseline. Secondary outcomes included score changes in the Alzheimer’s Disease Assessment Scale–Cognitive Subscale, Mini-Mental State Examination and Alzheimer’s Disease Cooperative Study–Activities of Daily Living scale. Moreover, single-pulse TMS in combination with EEG was used to assess neurophysiological changes in precuneus cortical excitability and oscillatory activity. Our findings show that patients that received precuneus repetitive magnetic stimulation presented a stable performance of the Clinical Dementia Rating Scale–Sum of Boxes score, whereas patients treated with sham showed a worsening of their score. Compared with the sham stimulation, patients in the precuneus stimulation group also showed also significantly better performances for the secondary outcome measures, including the Alzheimer’s Disease Assessment Scale–Cognitive Subscale, Mini-Mental State Examination and Alzheimer’s Disease Cooperative Study–Activities of Daily Living scale. Neurophysiological results showed that precuneus cortical excitability remained unchanged after 24 weeks in the precuneus stimulation group, whereas it was significantly reduced in the sham group. Finally, we found an enhancement of local gamma oscillations in the group treated with precuneus stimulation but not in patients treated with sham. We conclude that 24 weeks of precuneus rTMS may slow down cognitive and functional decline in Alzheimer’s disease. Repetitive TMS targeting the default mode network could represent a novel therapeutic approach in Alzheimer’s disease patients.

In Alzheimer disease (AD) animal models, synaptic dysfunction has recently been linked to a disorder of high-frequency neuronal activity. In patients, a clear relation between AD and oscillatory activity remains elusive. Here, we attempt to shed light on this relation by using a novel approach combining transcranial magnetic stimulation and electroencephalography (TMS-EEG) to probe oscillatory activity in specific hubs of the frontoparietal network in a sample of 60 mild-to-moderate AD patients.Sixty mild-to-moderate AD patients and 21 age-matched healthy volunteers (HVs) underwent 3 TMS-EEG sessions to assess cortical oscillations over the left dorsolateral prefrontal cortex, the precuneus, and the left posterior parietal cortex. To investigate the relations between oscillatory activity, cortical plasticity, and cognitive decline, AD patients underwent a TMS-based neurophysiological characterization and a cognitive evaluation at baseline. The latter was repeated after 24 weeks to monitor clinical evolution.AD patients showed a significant reduction of frontal gamma activity as compared to age-matched HVs. In addition, AD patients with a more prominent decrease of frontal gamma activity showed a stronger impairment of long-term potentiation-like plasticity and a more pronounced cognitive decline at subsequent follow-up evaluation at 24 weeks.Our data provide novel evidence that frontal lobe gamma activity is dampened in AD patients. The current results point to the TMS-EEG approach as a promising technique to measure individual frontal gamma activity in patients with AD. This index could represent a useful biomarker to predict disease progression and to evaluate response to novel pharmacological therapies. ANN NEUROL 2022;92:464-475.

Background: Neuroinflammatory cytokines can play a pivotal role in Alzheimer's disease (AD) contributing to the evolution of degenerative processes. Objective: We aimed at evaluating the levels of cerebrospinal fluid (CSF) inflammatory cytokines, chemokines, and growth factors in subjects with diagnosis of amnestic mild cognitive impairment and mild AD. Methods: We evaluated CSF contents of inflammatory cytokines in 66 patients divided according to the NIA-AA research framework and the APOE genotype. CSF of a group of cognitively unimpaired individuals (n = 23) was evaluated as control. All patients were evaluated for 24 months using Mini-Mental State Examination (MMSE). Results: We found significant increased levels of IL-4, IL-6, IL-8, and G-CSF in the CSF of A+/T-APOE4 carriers, respect to A+/T-patients homozygous for APOE3, respect to A+/T+ patients, regardless the APOE status, and respect to controls. Over a period of 24 months, A+/T-APOE4 carriers, with increased levels of cytokines, showed a preserved cognitive evaluation when compared to the other subgroups of patients (delta MMSE at 24 months respect to baseline: 0.10±0.35; p < 0.05). Conclusion: Our data suggest that during early phases of AD, in APOE4 carriers, Aβ pathology likely induces a specific cytokines pattern synthesis associated to cognitive preservation. These data highlight the different role that neuroinflammation can play in AD pathology based on the presence of specific CSF biomarkers and on the APOE status.

Background: Frontotemporal dementia (FTD) is a presenile neurodegenerative disease for which there is no effective pharmacological treatment. Recently, a link has been proposed between neuroinflammation and FTD. Objective: Here, we aim to investigate the effects of palmitoylethanolamide (PEA) combined with luteoline (PEA-LUT), an endocannabinoid with anti-inflammatory and neuroprotective effects, on behavior, cognition, and cortical activity in a sample of FTD patients. Methods: Seventeen patients with a diagnosis of probable FTD were enrolled. Cognitive and neurophysiological evaluations were performed at baseline and after 4 weeks of PEA-LUT 700 mg×2/day. Cognitive effects were assessed by Neuropsychiatric Inventory (NPI), Mini-Mental State Examination, Frontal Assessment Battery (FAB), Screening for Aphasia in Neurodegeneration, Activities of Daily Living-Instrumental Activities of Daily Living, and Frontotemporal Lobar Degeneration-modified Clinical Dementia Rating scale. To investigate in vivo neurophysiological effects of PEA-LUT, we used repetitive and paired-pulse transcranial magnetic stimulation (TMS) protocols assessing LTP-like cortical plasticity, short-interval intracortical inhibition, long-interval intracortical inhibition (LICI), and short-latency afferent inhibition. Moreover, we used TMS combined with EEG to evaluate the effects on frontal lobe cortical oscillatory activity. Results: Treatment with PEA-LUT was associated with an improvement in NPI and FAB scores. Neurophysiological evaluation showed a restoration of LICI, in particular at ISI 100 ms, suggesting a modulation of GABA(B) activity. TMS-EEG showed a remarkable increase of TMS-evoked frontal lobe activity and of high-frequency oscillations in the beta/gamma range. Conclusion: PEA-LUT could reduce behavioral disturbances and improve frontal lobe functions in FTD patients through the modulation of cortical oscillatory activity and GABA(B)ergic transmission. Keywords: Brain inflammation; EEG; GABA activity; behavioral symptoms; executive functions; frontotemporal dementia; transcranial magnetic stimulation.

The cerebellum is strongly implicated in learning new motor skills. Theta burst stimulation (TBS), a form of repetitive transcranial magnetic stimulation, can be used to influence cerebellar activity. Our aim was to explore the potential of cerebellar TBS in modulating visuo-motor adaptation, a form of motor learning, in young healthy subjects. Cerebellar TBS was applied immediately before the learning phase of a visuo-motor adaptation task (VAT), in two different experiments. Firstly, we evaluated the behavioral effects of continuous (cTBS), intermittent (iTBS) or sham TBS on the learning, re-adaptation and de-adaptation phases of VAT. Subsequently, we investigated the changes induced by iTBS or sham TBS on motor cortical activity related to each phase of VAT, as measured by concomitant TMS/EEG recordings. We found that cerebellar TBS induced a robust bidirectional modulation of the VAT performance. More specifically, cerebellar iTBS accelerated visuo-motor adaptation, by speeding up error reduction in response to a novel perturbation. This gain of function was still maintained when the novel acquired motor plan was tested during a subsequent phase of re-adaptation. On the other hand, cerebellar cTBS induced the opposite effect, slowing the rate of error reduction in both learning and re-adaptation phases. Additionally, TMS/EEG recordings showed that cerebellar iTBS induced specific changes of cortical activity in the interconnected motor networks. The improved performance was accompanied by an increase of TMS-evoked cortical activity and a generalized desynchronization of TMS-evoked cortical oscillations. Taken together, our behavioral and neurophysiological findings provide the first-time multimodal evidence of the potential efficacy of cerebellar TBS in improving motor learning, by promoting successful cerebellar-cortical reorganization.

Background Mechanisms of cortical plasticity have been recently investigated in Alzheimer's disease (AD) patients with transcranial magnetic stimulation protocols showing a clear impairment of long-term potentiation (LTP) cortical-like plasticity mechanisms. Objective We aimed to investigate mechanisms of cortico-cortical spike-timing dependent plasticity (STDP) in AD patients investigating the connections between posterior parietal cortex (PPC) and primary motor cortex (M1). Methods We used a cortico-cortical paired associative stimulation (cc-PAS) protocol to repeatedly activate the connection between PPC and M1 of the left-dominant hemisphere in a sample of fifteen AD patients and ten age-matched healthy subjects. PPC transcranial magnetic stimulation preceded (ccPAS +5) or followed M1 stimulation (ccPAS - 5) by 5 ms. Motor-evoked potentials (MEPs) were collected to assess the time course of the after effects of cc-PAS protocol measuring MEP amplitude as index of cortico-cortical associative plasticity. Results In healthy subjects, ccPAS - 5 protocol induced the expected long-lasting increase of MEP amplitude compatible with LTP-like cortical plasticity while PAS +5 protocol induced the opposite effect. AD patients did not show any significant modification of the amplitude of MEP after both ccPAS protocols. Conclusions Our study shows that in AD patients the time-locked activation of human cortico-cortical connections is not able to form STDP, reflecting an impairment of a multi-factor plasticity process.

OBJECTIVE Alzheimer's disease (AD) is considered an age-related disorder. However, it is unclear whether AD induces the same pathological and neurophysiological modifications in synaptic functions independently from age of disease onset. We used transcranial magnetic stimulation tools to investigate the mechanisms of cortical plasticity and sensory-motor integration in AD patients with a wide range of disease onset. METHODS We evaluated newly diagnosed sporadic AD (n = 54) in comparison with healthy age-matched controls (HS; n = 24). Cortical plasticity mechanisms of long-term potentiation (LTP) or of long-term depression (LTD) were assessed using respectively intermittent (iTBS) or continuous theta burst stimulation (cTBS) protocols. Sensory-motor integration was evaluated by means of short afferent inhibition (SAI) protocol. RESULTS AD patients show after iTBS an impairment of LTP-like cortical plasticity forming a paradoxical LTD in comparison to HS. LTD-like cortical plasticity is similar between AD and HS. LTP-like cortical plasticity is not associated with age, but AD patients presenting with more altered LTP-like cortical plasticity have more-severe cognitive decline at 18 months. SAI is impaired in AD and shows a strong association with the individual age of subjects rather than with disease age of onset. INTERPRETATION Cortical LTP disruption is a central mechanism of AD that is independent from age of onset. AD can be described primarily as a disorder of LTP-like cortical plasticity not influenced by physiological aging and associated with a more-severe cognitive decline. Ann Neurol 2016;80:202-210.

Background Alzheimer's disease (AD) is characterized by a primary impairment of long-term declarative memory caused by deposition of misfolded protein aggregates. Experimental studies showed that AD neuropathological alterations impair synaptic plasticity and memory performance. Transcranial Magnetic Stimulation protocols have been recently introduced to investigate altered mechanisms of cortical plasticity in AD patients. Aim To investigate relationship between Long-Term Potentiation (LTP)-like cortical plasticity and patients’ neuropsychological performance. Methods We applied intermittent theta burst stimulation and extensive neuropshycological battery in 75 newly diagnosed AD patients. Results We found that LTP-like cortical plasticity impairment is selectively associated to a less efficient verbal memory (r = 0.45; p = 0.002), but not to other cognitive functions, independently from biomarkers and other demographic and clinical factors. Conclusion These findings suggest that LTP-like cortical plasticity may represent a neurophysiological surrogate of memory in AD patients by evaluating the weight of pathological changes responsible for cognitive dysfunction.

Memory loss is one of the first symptoms of typical Alzheimer's disease (AD), for which there are no effective therapies available. The precuneus (PC) has been recently emphasized as a key area for the memory impairment observed in early AD, likely due to disconnection mechanisms within large-scale networks such as the default mode network (DMN). Using a multimodal approach we investigated in a two-week, randomized, sham-controlled, double-blinded trial the effects of high-frequency repetitive transcranial magnetic stimulation (rTMS) of the PC on cognition, as measured by the Alzheimer Disease Cooperative Study Preclinical Alzheimer Cognitive Composite in 14 patients with early AD (7 females). TMS combined with electroencephalography (TMS-EEG) was used to detect changes in brain connectivity. We found that rTMS of the PC induced a selective improvement in episodic memory, but not in other cognitive domains. Analysis of TMS-EEG signal revealed an increase of neural activity in patients' PC, an enhancement of brain oscillations in the beta band and a modification of functional connections between the PC and medial frontal areas within the DMN. Our findings show that high-frequency rTMS of the PC is a promising, non-invasive treatment for memory dysfunction in patients at early stages of AD. This clinical improvement is accompanied by modulation of brain connectivity, consistently with the pathophysiological model of brain disconnection in AD.

BackgroundFew data exist on malignant middle cerebral artery infarction (MMI) among patients with acute ischemic stroke (AIS) after endovascular treatment (ET). Numerous predictors of MMI evolution have been proposed, but a comprehensive research of patients undergoing ET has never been performed. Our purpose was to find a practical model to determine robust predictors of MMI in patients undergoing ET.MethodsPatients from a prospective single-center database with AIS secondary to large intracranial vessel occlusion of the anterior circulation, treated with ET, were retrospectively analyzed. We investigated demographic, clinical, and radiological data. Multivariate regression analysis was used to identify clinical and imaging predictors of MMI.Results98 patients were included in the analysis, 35 of whom developed MMI (35.7%). No differences in the rate of successful reperfusion and time from stroke onset to reperfusion were found between the MMI and non-MMI groups. The following parameters were identified as independent predictors of MMI: systolic blood pressure (SBP) on admission (p=0.008), blood glucose (BG) on admission (p=0.024), and the CTangiography (CTA) Alberta Stroke Program Early CT Score (ASPECTS) (p=0.001). A scoreof ≤5 in CTA ASPECTS was the best cut-off to predict MMI evolution (sensitivity 46%; specificity 97%; positive predictive value 78%; negative predictive value 65%).Conclusionsin our study a clinical and radiological features-based model was strongly predictive of MMI evolution in AIS. High SBP and BG on admission and, especially, a CTA ASPECTS ≤5 may help to make decisions quickly, regardless of time to treatment and successful reperfusion.

Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by persistent inflammation orchestrated by cluster of differentiation (CD) 4 T helper (Th) cells. In particular, Th1 and Th17 cells amplify, whereas T regulatory (Treg) cells moderate inflammation. The role of other Th subsets in MS is not clear. In the present study, we investigated the generation of different Th responses by human dendritic cells (DCs) in MS. We compared the production of several Th cytokines by naive CD4+ T-cells polarized with myeloid and plasmacytoid DCs (mDCs and pDCs) in healthy donors (HD) and relapsing–remitting (RR)-MS patients. We found that resiquimod-stimulated mDCs were able to activate Th17 differentiation, whereas pDCs induced interleukin (IL)-10-producing Th cells. Surprisingly, resiquimod-stimulated pDCs from MS patients also significantly induced the differentiation of Th9 cells, which produce IL-9 and are known to be involved in allergic diseases. We investigated the potential role of IL-9 in MS. We found that IL-9 activated signal transducer and activator of transcription (STAT) 1 and STAT5 phosphorylation and interfered with IL-17 and interferon (IFN) regulatory transcription factor (IRF)-4 expression in Th17-polarized cells. Moreover, in the cerebrospinal fluid (CSF) of 107 RR-MS patients, IL-9 inversely correlated with indexes of inflammatory activity, neurodegeneration and disability progression of MS. High levels of IL-9 were associated with the absence of IL-17 in the CSF of RR-MS patients. Our results demonstrate a Th9-inducing potential of pDCs in MS, suggesting an immunoregulatory role leading to attenuation of the exaggerated Th17 inflammatory response.

Background: Subtle diffuse intrathecal inflammation is undetectable by conventional neuroimaging, and could influence multiple sclerosis (MS) disease course. Objective: To explore the role of subclinical persisting intrathecal inflammation in radiologically isolated syndrome (RIS) or clinically isolated syndrome (CIS) conversion to MS, and in early MS disease reactivation. Methods: One-hundred ninety-three subjects with RIS, CIS, relapsing–remitting (RR), or primary progressive (PP) MS were included, along with 76 matched controls. Cerebrospinal fluid (CSF) levels of interleukin-8 (IL-8), a major proinflammatory cytokine, were measured as a biomarker of intrathecal inflammation. Patients were followed up for 2 years. Clinical and imaging measures of disease progression were recorded. Results: High central contents of IL-8 were associated to clinical progression in subjects with RIS, and to the risk of conversion to MS in subjects with CIS. Asymptomatic intrathecal inflammation placed subjects at risk for MS conversion, even regardless lesion load. CSF IL-8 levels were higher in RR MS with high disease activity. Higher number of relapses in the first two years since diagnosis and shorter first inter-attack intervals were observed in patients with high levels of IL-8. Conclusion: IL-8 might provide utility in determining the presence of active intrathecal inflammation, and could be important in diagnostically undefined cases.

In Alzheimer’s disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau.

Background Sympathovagal imbalance has been associated with poor prognosis in chronic diseases, but there is conflicting evidence in multiple sclerosis. Objectives The objective of this study was to investigate the autonomic nervous system dysfunction correlation with inflammation and progression in multiple sclerosis. Methods Heart rate variability was analysed in 120 multiple sclerosis patients and 60 healthy controls during supine rest and head-up tilt test; the normalised units of low frequency and high frequency power were considered to assess sympathetic and vagal components, respectively. Correlation analyses with clinical and radiological markers of disease activity and progression were performed. Results Sympathetic dysfunction was closely related to the progression of disability in multiple sclerosis: progressive patients showed altered heart rate variability with respect to healthy controls and relapsing–remitting patients, with higher rest low frequency power and lacking the expected low frequency power increase during the head-up tilt test. In relapsing–remitting patients, disease activity, even subclinical, was associated with lower rest low frequency power, whereas stable relapsing–remitting patients did not differ from healthy controls. Less sympathetic reactivity and higher low frequency power at rest were associated with incomplete recovery from relapse. Conclusions Autonomic balance appears to be intimately linked with both the inflammatory activity of multiple sclerosis, which is featured by an overall hypoactivity of the sympathetic nervous system, and its compensatory plastic processes, which appear inefficient in case of worsening and progressive multiple sclerosis.

Objectives To evaluate the safety and efficacy of endovascular therapy in elderly adults treated for acute ischemic stroke. Design Retrospective cohort study. Setting Comprehensive Stroke Center, University of Tor Vergata, Rome, Italy. Participants Elderly adults treated for acute ischemic stroke (N = 219). Measurements Participants were divided into two groups based on their age (n = 62, ≥80; n = 157, .99) or symptomatic (11% vs 14%, P = .6) ICH, or mortality (40.3% vs 29.2%, P = .14). Multivariable analysis revealed that, in the older group, onset National Institute of Health Stroke Scale (NIHSS) score (odds ratio (OR) = 0.65, 95% confidence interval (CI) = 0.44–0.96, P = .03) and 24-hour clinical improvement (OR = 141.13, 95% CI = 2.96–6,720.7, P = .01) were independent predictors of 3-month functional independence. Conclusion These findings suggest that endovascular treatment for stroke in selected elderly adults could be safe and effective. Major determinants of outcome in this subgroup of elderly patients are presentation NIHSS score and 24-hour clinical improvement.

BACKGROUND AND PURPOSE: Noncontrast CT ASPECTS has been investigated as a predictor of outcome in patients with acute ischemic stroke. Our purpose was to investigate whether CTA source images are a better predictor of clinical and radiologic outcomes than NCCT ASPECTS in candidates for endovascular stroke therapy. MATERIALS AND METHODS: CT scans of patients (n = 124) were independently evaluated by 2 readers for baseline NCCT and CTA source image ASPECTS and for follow-up ASPECTS. An mRS of ≤2 at 3 months was considered a favorable outcome. Receiver operating characteristic curve analysis was used to assess the ability of NCCT and CTA source image ASPECTS to identify patients with favorable outcomes. A stepwise multiple regression analysis was performed to find independent predictors of outcome. RESULTS: Baseline CTA source image ASPECTS correlated better than NCCT ASPECTS with follow-up ASPECTS (r = 0.76 versus r = 0.51; P for comparison of the 2 coefficients < .001). Receiver operating characteristic curve analysis showed that baseline CTA source image ASPECTS compared with NCCT ASPECTS can better identify patients with favorable outcome (CTA source image area under the curve = 0.83; 95% CI, 0.76–0.91; NCCT area under the curve = 0.67; 95% CI, 0.58–0.77; P < .001). Finally, the stepwise regression analysis showed that lower age, good recanalization, lower time to recanalization, and good baseline CTA source image ASPECTS, not NCCT ASPECTS, were independent predictors of favorable outcome. CONCLUSIONS: CTA source image ASPECTS predicts outcome better than NCCT ASPECTS; this finding suggests CTA rather than NCCT as a main step in the decision-making process for patients with acute ischemic stroke.

During multiple sclerosis (MS) inflammatory attacks, and in subsequent clinical recovery phases, immune cells contribute to neuronal and oligodendroglial cell survival and tissue repair by secreting growth factors. Animal studies showed that growth factors also play a substantial role in regulating synaptic plasticity, and namely in long-term potentiation (LTP). LTP could drive clinical recovery in relapsing patients by restoring the excitability of denervated neurons. We recently reported that maintenance of synaptic plasticity reserve is crucial to contrast clinical deterioration in MS and that the platelet-derived growth factor (PDGF) may play a key role in its regulation. We also reported that a Hebbian form of LTP-like cortical plasticity, explored by paired associative stimulation (PAS), correlates with clinical recovery from a relapse in MS. Here, we explored the role of PDGF in clinical recovery and in adaptive neuroplasticity in relapsing-remitting MS (RR-MS) patients. We found a correlation between the cerebrospinal fluid (CSF) PDGF concentrations and the extent of clinical recovery after a relapse, as full recovery was more likely observed in patients with high PDGF concentrations and poor recovery in subjects with low PDGF levels. Consistently with the idea that PDGF-driven synaptic plasticity contributes to attenuate the clinical consequences of tissue damage in RR-MS, we also found a striking correlation between CSF levels of PDGF and the amplitude of LTP-like cortical plasticity explored by PAS. CSF levels of fibroblast growth factor, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor did not correlate with clinical recovery nor with measures of synaptic transmission and plasticity.

Background: Chronic inflammation leads to gray matter damage in progressive multiple sclerosis (MS), but the mechanism linking inflammation and neurodegeneration is unclear. Objective: The objective of this paper is to investigate the synaptic mechanism of inflammatory neurodegeneration in progressive forms of MS. Methods: Cytokine and neurofilament-light were determined in cerebrospinal fluid (CSF) of MS patients. In vitro electrophysiology and cell swelling experiments were performed to measure the effects of inflammatory cytokines in the CSF of MS patients on synaptic transmission and neuronal integrity. Results: Tumor necrosis factor-α (TNF) was higher in CSF of progressive MS subjects, and caused excitotoxic neuronal death in vitro. In murine brain slices incubated in the presence of CSF from progressive MS, in fact, we observed increased spontaneous excitatory postsynaptic currents (sEPSCs) and glutamate-mediated neuronal swelling through a mechanism dependent on enhanced TNF signaling. We also suggested a pathogenic role of B cells in TNF CSF increase, exacerbation of glutamatergic transmission and neuronal damage, since CNS depletion of B cells with intrathecal rituximab caused a dramatic reduction of TNF levels, of TNF-induced sEPSC alterations, and of neurofilament CSF concentrations in a patient with progressive MS. Conclusion: Our results point to TNF as a primary neurotoxic molecule in progressive forms of MS.

Objective:To explore the inflammatory processes in the pathogenesis of psychiatric symptoms and the prognostic value of psychiatric comorbidities in multiple sclerosis (MS).Methods:Four hundred five patients with relapsing-remitting (RR) MS underwent psychiatric evaluation by means of Beck Depression Inventory II (BDI-II) and State/Trait Anxiety Inventory (STAI-Y). The inflammatory activity level was assessed by MRI. In a subset of 111 treatment-naive patients, CSF levels of proinflammatory cytokines were determined. Correlation and regression analyses were performed to determine associations between variables.Results:Relapsing patients demonstrated greater values of STAI-state and BDI-II compared with remitting patients but comparable trait-anxiety scores. There were no significant differences in psychometric parameters between relapsing and asymptomatic MRI-active patients, highlighting the effect of subclinical inflammation on mood disturbances. A significant reduction of STAI-state and BDI-II scores was recorded, along with the subsiding of neuroinflammation. Interleukin-2 CSF levels were found to correlate with STAI-state, while tumor necrosis factor-α and interleukin-1β correlated with BDI-II. Because emotional disorders were associated with subclinical inflammation, variations of the psychometric profile were able to detect subclinical reactivation earlier. In line with this, high STAI-state values considerably predicted the possibility of disease reactivation.Conclusions:Mood alterations are induced by intrathecal inflammation, even though not clinically apparent, and are able to predict inflammatory reactivations in RRMS. Inflammation is therefore a biological event, not less important than the traditional psychosocial factors, involved in mood disorders.

Background Flu-like syndrome (FLS) is a common adverse event experienced by patients with relapsing-remitting multiple sclerosis (RRMS) treated with interferon beta (IFNβ). FLS can lead to poor treatment adherence and early IFNβ discontinuation. The involvement of interleukin-6 (IL-6) in the occurrence of FLS has been suggested. We hypothesized that cetirizine, a second-generation histamine H1 receptor antagonist able to reduce the levels of IL-6, might improve IFNβ-induced FLS. Methods We conducted a pilot, cross-over, randomized, placebo-controlled, double-blind study to evaluate the efficacy of cetirizine 10 mg added after each IFNβ injection to the standard of care for FLS (acetaminophen or nonsteroidal anti-inflammatory drugs) on FLS in patients with RRMS treated with IFNβ. Patients were randomized to two treatment sequences: 1) 4-week treatment with placebo added to the standard treatment for FLS, followed by 4-week treatment with cetirizine added to the standard of care, and 2) first addition of cetirizine, then of placebo. The primary efficacy endpoint was the mean change of FLS severity [11-point visual analog scale (VAS)] after 4 weeks of treatment within each sequence. Results Forty-five patients (71.1% female, mean age 39.1 years, mean time from RRMS diagnosis 5.8 years) were randomized to treatment sequences 1 and 2. The differences between cetirizine and placebo in the intensity of FLS were not statistically significant: total mean VAS scores at 4 hours from IFNβ injection were 3.57 and 3.42 for cetirizine and placebo, respectively (difference -0.15; 95% confidence interval: from -0.74 to 0.44; p = 0.6029). The two treatments were similar also with regard to other efficacy measures considered and to the safety/tolerability profile. Conclusions The addition of cetirizine to the standard of care for IFNβ-induced FLS in patients with RRMS does not seem to provide significant benefits compared with placebo. Further effort is required to understand the mechanisms underlying IFNβ-induced FLS. Trial registration EudraCT 2013-001055-12.

Background Multiple Sclerosis (MS) is a widespread progressive neurologic disease with consequent impairments in daily activities. Disorders of balance are frequent and equilibrium tests are potentially useful to quantify disability and to verify treatment effectiveness. The fair sensitivity of the widely used not-perturbed tests to detect balance disturbances in MS patients have prompted the development of mechatronic systems capable to impose known equilibrium perturbations, in order to challenge the balance control and, consequently, to better assess the level of impairment. We sought to clarify whether the proposed perturbed-test is capable to discriminate healthy subjects from patients with MS, even in mild or in the absence of clinically evident balance disturbances. Methods We assessed balance performances of 17 adults with MS and 13 age-matched healthy controls (HC) using both perturbed (PT) and not-perturbed (NPT) postural tests by means of a 3 Degree Of Freedom (DOF) rotational mechatronic platform. Participants stood barefoot on the platform in standing position and their center of pressure (CoP) was gathered by using a pressure matrix. Each trial lasted 30 s and was carried out with and without visual stimuli. Several postural indices were computed for each trial. Correlations between postural indices and clinical scales were analyzed. Results No significant differences were found between groups for all indices when subjects performed NPTs. Conversely, significant differences in postural indices between MS and HC emerged during PTs. Additionally, PTs revealed significant differences between patients without any cerebellar impairment (cerebellar EDSS subscore equal to 0) and HC. The discrimination capability of PTs was confirmed by the ROC analysis. No significant change of the selected metrics occurred in HC when NPTs were performed with eyes closed, while indices presented a significant worsening in MS subjects. Conclusions Not-perturbed tests showed lower sensitivity than perturbed ones in the identification of equilibrium impairments in minimally disabled MS patients. However, not-perturbed tests allow to better evaluate the influence of visual flow disturbances on balance control in MS. In conclusion, our findings proved that the use of the novel tests based on a 3DOF mechatronic device represents an effective tool to investigate early balance disturbances in MS.

Ischemic stroke is the leading cause of disability, but effective therapies are currently widely lacking. Recovery from stroke is very much dependent on the possibility to develop treatments able to both halt the neurodegenerative process as well as to foster adaptive tissue plasticity. Here we show that ischemic mice treated with neural precursor cell (NPC) transplantation had on neurophysiological analysis, early after treatment, reduced presynaptic release of glutamate within the ipsilesional corticospinal tract (CST), and an enhanced NMDA-mediated excitatory transmission in the contralesional CST. Concurrently, NPC-treated mice displayed a reduced CST degeneration, increased axonal rewiring, and augmented dendritic arborization, resulting in long-term functional amelioration persisting up to 60 d after ischemia. The enhanced functional and structural plasticity relied on the capacity of transplanted NPCs to localize in the peri-ischemic and ischemic area, to promote the upregulation of the glial glutamate transporter 1 (GLT-1) on astrocytes and to reduce peri-ischemic extracellular glutamate. The upregulation of GLT-1 induced by transplanted NPCs was found to rely on the secretion of VEGF by NPCs. Blocking VEGF during the first week after stroke reduced GLT-1 upregulation as well as long-term behavioral recovery in NPC-treated mice. Our results show that NPC transplantation, by modulating the excitatory–inhibitory balance and stroke microenvironment, is a promising therapy to ameliorate disability, to promote tissue recovery and plasticity processes after stroke.SIGNIFICANCE STATEMENTTissue damage and loss of function occurring after stroke can be constrained by fostering plasticity processes of the brain. Over the past years, stem cell transplantation for repair of the CNS has received increasing interest, although underlying mechanism remain elusive. We here show that neural stem/precursor cell transplantation after ischemic stroke is able to foster axonal rewiring and dendritic plasticity and to induce long-term functional recovery. The observed therapeutic effect of neural precursor cells seems to underlie their capacity to upregulate the glial glutamate transporter on astrocytes through the vascular endothelial growth factor inducing favorable changes in the electrical and molecular stroke microenvironment. Cell-based approaches able to influence plasticity seem particularly suited to favor poststroke recovery.

Background The available clinical outcome measures of disability in multiple sclerosis are not adequately responsive or sensitive. Objective To investigate the feasibility of inertial sensor-based gait analysis in multiple sclerosis. Methods A cross-sectional study of 80 multiple sclerosis patients and 50 healthy controls was performed. Lower-limb kinematics was evaluated by using a commercially available magnetic inertial measurement unit system. Mean and standard deviation of range of motion (mROM, sROM) for each joint of lower limbs were calculated in one minute walking test. A motor performance index (E) defined as the sum of sROMs was proposed. Results We established two novel observer-independent measures of disability. Hip mROM was extremely sensitive in measuring lower limb motor impairment, being correlated with muscle strength and also altered in patients without clinically detectable disability. On the other hand, E index discriminated patients according to disability, being altered only in patients with moderate and severe disability, regardless of walking speed. It was strongly correlated with fatigue and patient-perceived health status. Conclusions Inertial sensor-based gait analysis is feasible and can detect clinical and subclinical disability in multiple sclerosis.

BACKGROUND AND PURPOSE Alterations of glutamate-mediated synaptic transmission occur early during neuroinflammatory insults, and lead to degenerative neuronal damage in multiple sclerosis (MS) and also in experimental autoimmune encephalomyelitis (EAE), which is a murine model of MS. Fingolimod is an effective orally active agent for the treatment of MS, affecting lymphocyte invasion of the brain. However, it is still unclear if fingolimod can be neuroprotective in this disorder. EXPERIMENTAL APPROACH Using neurophysiological recordings and morphological evaluation of dendritic integrity, we evaluated the effects of oral fingolimod on the clinical score of EAE mice in order to determine whether the compound was associated with preservation of synaptic transmission. KEY RESULTS Oral fingolimod prevented and reversed the pre- and postsynaptic alterations of glutamate transmission in EAE mice. These effects were associated with a clear amelioration of the clinical deterioration seen in EAE mice, and with a significant inhibition of neuronal dendritic pathology. Fingolimod did not alter the spontaneous excitatory postsynaptic currents in control animals, suggesting that only the pathological processes behind the inflammation-induced defects in glutamate transmission were modulated by this compound. CONCLUSIONS AND IMPLICATIONS The beneficial effects of fingolimod on the clinical, synaptic and dendritic abnormalities of murine EAE might correlate with the neuroprotective actions of this agent, as observed in MS patients. LINKED ARTICLE This article is commented on by Gillingwater, pp. 858–860 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01612.x

Objective: The frequency of inflammatory episodes in the early stages of multiple sclerosis (MS) has been correlated with late neurodegeneration, but the mechanism by which inflammation gives rise to delayed neuronal damage is unknown. Increased activity of the neurotransmitter glutamate is thought to play a role in the inflammation-driven neurodegenerative process of MS, and therefore we tested whether inflammatory cytokines released during acute MS attacks have the property of enhancing glutamate-mediated transmission and excitotoxicity in central neurons. Methods: We compared the effect of cerebrospinal fluid (CSF) from active and quiescent MS patients on glutamate-mediated excitatory postsynaptic currents (EPSCs) and excitotoxic damage in rodent brain slices. We also measured CSF concentrations of tumor necrosis factor-α, of interleukin-1β (IL-1β), and of IL-1 receptor antagonist (IL-1ra), and correlated cytokine levels with cortical excitability assessed in MS patients by means of paired-pulse transcranial magnetic stimulation (TMS). Results: CSF from MS patients with enhanced brain lesions at magnetic resonance imaging was able to increase spontaneous EPSC frequency and glutamate-mediated neuronal swelling in vitro, through a mechanism dependent on enhanced IL-1β signaling and increased glutamate α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor stimulation. Furthermore, IL-1β/IL-1ra ratio was significantly higher in the CSF of active MS subjects, and correlated with intracortical facilitation, an accredited TMS measure of glutamate transmission. Finally, we identified for the first time transient receptor potential vanilloid 1 channels as essential intermediates for the synaptic action of IL-1β on central glutamatergic synapses. Interpretation: Our results provide compelling evidence of the synaptic mechanism linking inflammation and excitotoxic neurodegeneration in MS. ANN NEUROL 2012;71:76–83

Dimethyl fumarate (DMF), recently approved as an oral immunomodulatory treatment for relapsing-remitting multiple sclerosis (MS), metabolizes to monomethyl fumarate (MMF) which crosses the blood–brain barrier and has demonstrated neuroprotective effects in experimental studies. We postulated that MMF exerts neuroprotective effects through modulation of microglia activation, a critical component of the neuroinflammatory cascade that occurs in neurodegenerative diseases such as MS. To ascertain our hypothesis and define the mechanistic pathways involved in the modulating effect of fumarates, we used real-time PCR and biochemical assays to assess changes in the molecular and functional phenotype of microglia, quantitative Western blotting to monitor activation of postulated pathway components, and ex vivo whole-cell patch clamp recording of excitatory post-synaptic currents in corticostriatal slices from mice with experimental autoimmune encephalomyelitis (EAE), a model for MS, to study synaptic transmission. We show that exposure to MMF switches the molecular and functional phenotype of activated microglia from classically activated, pro-inflammatory type to alternatively activated, neuroprotective one, through activation of the hydroxycarboxylic acid receptor 2 (HCAR2). We validate a downstream pathway mediated through the AMPK–Sirt1 axis resulting in deacetylation, and thereby inhibition, of NF-κB and, consequently, of secretion of pro-inflammatory molecules. We demonstrate through ex vivo monitoring of spontaneous glutamate-mediated excitatory post-synaptic currents of single neurons in corticostriatal slices from EAE mice that the neuroprotective effect of DMF was exerted on neurons at pre-synaptic terminals by modulating glutamate release. By exposing control slices to untreated and MMF-treated activated microglia, we confirm the modulating effect of MMF on microglia function and, thereby, its indirect neuroprotective effect at post-synaptic level. These findings, whereby DMF-induced activation of a new HCAR2-dependent pathway on microglia leads to the modulation of neuroinflammation and restores synaptic alterations occurring in EAE, represent a possible novel mechanism of action for DMF in MS. Electronic supplementary material The online version of this article (doi:10.1007/s00401-015-1422-3) contains supplementary material, which is available to authorized users.

Background: Acute optic neuritis is often in association with multiple sclerosis (MS). Proinflammatory cytokines trigger neuronal damage in neuroinflammatory disorders but their role in optic neuritis is poorly investigated. Objective: The objective of this work is to investigate the associations of intrathecal contents of proinflammatory cytokines with transient and persistent dysfunctions after optic neuritis. Methods: In 50 MS patients followed for up to six months, cerebrospinal fluid (CSF) levels of IL-1β, TNF and IL-8 were determined, along with clinical, neurophysiological and morphological measures of optic neuritis severity. Results: Visual impairment, measured by high- and low-contrast visual acuity, and delayed visual-evoked potential (VEP) latencies were significantly correlated to IL-8 levels during optic neuritis. IL-8 at the time of optic neuritis was also associated with persistent demyelination and final axonal loss, inferred by VEP and optical coherence tomography measures, respectively. Contents of IL-8 were correlated to functional visual outcomes, being higher among patients with incomplete recovery. Multivariate analysis confirmed that IL-8 significantly predicted final visual acuity, at equal values of demographics and baseline visual scores. Conclusion: Our study points to IL-8 as the main inflammatory cytokine associated with demyelination and secondary neurodegeneration in the optic nerve after optic neuritis.

nized with PLP139-151, a clearly defined relapsing–remitting disease course similar to relapsing–remitting multiple sclerosis. OLC populations in the CNS were quantified by flow cytometry throughout an EAE disease course. To prevent the large infiltration of immune cells from skewing resident CNS cell analysis, we incorporated an antibody against the hematopoietic marker CD45 into the panel and gated out those cells in OLC analysis. Flow cytometry results demonstrated a robust loss of oligodendrocytes during onset of EAE and an early expansion of OPCs. By disease peak the early OPCs expressed more mature markers of OLCs but never fully differentiated into mature myelin-forming oligodendrocytes throughout the rest of the disease course. The early expansion of OPCs was further associated with an increase in the chemotactic protein CD44 in OPCs from the brain. In summary we present here quantification of OLCs and functional global analysis of the myelinproducing cells in EAE mice by flow cytometry. Characterization in mouse models of human CNS disease presents a rapid means to evaluate potential therapeutic interventions in vivo for direct and indirect effects on demyelination and myelin regeneration.

Summary Aim Natalizumab (NTZ) discontinuation leads to multiple sclerosis (MS) recurrence, but represents the only known strategy to limit the risk of progressive multifocal leukoencephalopathy (PML) in JCV seropositive patients. Here, we compared the clinical and imaging features of three groups of patients who discontinued NTZ treatment. Methods We treated 25 patients with subcutaneous INFβ-1b (INF group), 40 patients with glatiramer acetate (GA group), and 40 patients with GA plus pulse steroid (GA+CS group). Results Six of 25 patients (24%) of the INF group were relapse-free 6 months after NTZ suspension. In GA group, a significant higher proportion of patients (26 of 40 patients, 65%) were relapse-free (P

Background Absence of clinical and radiological activity in relapsing–remitting multiple sclerosis (RRMS) is perceived as disease remission. We explored the role of persisting inflammation during remission in disease evolution. Methods Cerebrospinal fluid (CSF) levels of interleukin 1β (IL-1β), a major proinflammatory cytokine, were measured in 170 RRMS patients at the time of clinical and radiological remission. These patients were then followed up for at least 4 years, and clinical, magnetic resonance imaging (MRI) and optical coherence tomography (OCT) measures of disease progression were recorded. Results Median follow-up of RRMS patients was 5 years. Detection of CSF IL-1β levels at the time of remission did not predict earlier relapse or new MRI lesion formation. Detection of IL-1β in the CSF was instead associated with higher progression index (PI) and Multiple Sclerosis Severity Scale (MSSS) scores at follow-up, and the number of patients with sustained Expanded Disability Status Scale (EDSS) or Multiple Sclerosis Functional Composite worsening at follow-up was higher in individuals with detectable levels of IL-1β. Patients with undetectable IL-1β in the CSF had significantly lower PI and MSSS scores and a higher probability of having a benign MS phenotype. Furthermore, patients with undetectable CSF levels of IL-1β had less retinal nerve fiber layer thickness and macular volume alterations visualized by OCT compared to patients with detectable IL-1β. Conclusions Our results suggest that persistence of a proinflammatory environment in RRMS patients during clinical and radiological remission influences midterm disease progression. Detection of IL-1β in the CSF at the time of remission appears to be a potential negative prognostic factor in RRMS patients.

Background Understanding how inflammation causes neuronal damage is of paramount importance in multiple sclerosis (MS) and in other neurodegenerative diseases. Here we addressed the role of the apoptotic cascade in the synaptic abnormalities and neuronal loss caused by the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor (TNF-α) in brain tissues, and disease progression caused by inflammation in relapsing-remitting MS (RRMS) patients. Results The effect of IL-1β, but not of TNF-α, on glutamate-mediated excitatory postsynaptic currents was blocked by pifithrin-α (PFT), inhibitor of p53. The protein kinase C (PKC)/transient receptor potential vanilloid 1 (TRPV1) pathway was involved in IL-1β-p53 interaction at glutamatergic synapses, as pharmacological modulation of this inflammation-relevant molecular pathway affected PFT effects on the synaptic action of IL-1β. IL-1β-induced neuronal swelling was also blocked by PFT, and IL-1β increased the expression of p21, a canonical downstream target of activated p53. Consistent with these in vitro results, the Pro/Pro genotype of p53, associated with low efficiency of transcription of p53-regulated genes, abrogated the association between IL-1β cerebrospinal fluid (CSF) levels and disability progression in RRMS patients. The interaction between p53 and CSF IL-1β was also evaluated at the optical coherence tomography (OCT), showing that IL-1β-driven neurodegenerative damage, causing alterations of macular volume and of retinal nerve fibre layer thickness, was modulated by the p53 genotype. Conclusions Inflammatory synaptopathy and neurodegeneration caused by IL-1β in RRMS patients involve the apoptotic cascade. Targeting IL-1β-p53 interaction might result in significant neuroprotection in MS.

Neuroplasticity is essential to prevent clinical worsening despite continuing neuronal loss in several brain diseases, including multiple sclerosis (MS). The precise nature of the adaptation mechanisms taking place in MS brains, ensuring protection from disability appearance and accumulation, is however unknown. Here, we explored the hypothesis that long-term synaptic potentiation (LTP), potentially able to minimize the effects of neuronal loss by providing extra excitation of denervated neurons, is the most relevant form of adaptive plasticity in stable MS patients, and it is disrupted in progressing MS patients. We found that LTP, explored by means of transcranial magnetic theta burst stimulation over the primary motor cortex, was still possible, and even favored, in stable relapsing-remitting (RR-MS) patients, whereas it was absent in individuals with primary progressive MS (PP-MS). We also provided evidence that platelet-derived growth factor (PDGF) plays a substantial role in favoring both LTP and brain reserve in MS patients, as this molecule: (1) was reduced in the CSF of PP-MS patients, (2) enhanced LTP emergence in hippocampal mouse brain slices, (3) was associated with more pronounced LTP in RR-MS patients, and (4) was associated with the clinical compensation of new brain lesion formation in RR-MS. Our results show that brain plasticity reserve, in the form of LTP, is crucial to contrast clinical deterioration in MS. Enhancing PDGF signaling might represent a valuable treatment option to maintain brain reserve and to attenuate the clinical consequences of neuronal damage in the progressive phases of MS and in other neurodegenerative disorders.