Tissue Doppler ultrasound of arm muscles to assess myotonia in myotonic dystrophies: An exploratory study.

INTRODUCTION/AIMS: Myotonia is a key symptom of myotonic dystrophies (DM), and its quantification is challenging. This exploratory study evaluated the utility of tissue Doppler ultrasound (TDU) to assess myotonia in DM. METHODS: Twelve DM patients (seven type-1 DM [DM1] and five type-2 DM [DM2]) and 20 age-matched healthy subjects were included in this cross-sectional study. After measuring cross-sectional areas of the flexor digitorum superficialis (FDS) and extensor digitorum communis (EDC) muscles in a resting state, muscle contraction/relaxation time, time to peak tissue velocity, peak tissue velocity and velocity gradients of these muscles were measured via TDU while performing forced fist unclenching after fist closure. Additionally, grip strength, Medical Research Council Sum score and patient-reported myotonia severity scores were assessed. RESULTS: DM1 and DM2 patients had a lower grip strength than healthy subjects (p = .0001/p = .002). Patient-reported myotonia did not differ between DM1 and DM2 patients. DM1 patients revealed FDS and EDC atrophy compared to DM2 patients and healthy subjects (p = .003/p = .004). TDU revealed prolonged muscle contraction and relaxation times in both DM subtypes, with prolonged time to reach FDS peak relaxation velocity and altered peak FDS relaxation velocity only in DM1 patients (p = .03/p = .003). Peak FDS relaxation velocity correlated inversely with C(C)TG repeat numbers in DM patients. Sensitivity of TDU parameters to detect myotonic dystrophy varied between 50% and 75%, with a specificity of 95%. DISCUSSION: Our exploratory study suggests that TDU could serve as a novel tool to quantify myotonia in DM patients, but larger follow-up studies are warranted to validate its diagnostic accuracy.

Immunomodulatory effects of intravenous and subcutaneous immunoglobulin in chronic inflammatory demyelinating polyneuropathy: An observational study.

BACKGROUND AND PURPOSE: It is not known whether the route of administration affects the mechanisms of action of therapeutic immunoglobulin in chronic inflammatory demyelinating polyneuropathy (CIDP). The aim of this study, therefore, was to compare the immunomodulatory effects of intravenous (IVIg) and subcutaneous immunoglobulin (SCIg) in patients with CIDP and in IVIg-treated common variable immunodeficiency (CVID) patients. METHODS: Serum and peripheral blood mononuclear cell samples were obtained from 30 CIDP patients receiving IVIg, 10 CIDP patients receiving SCIg, and 15 patients with CVID receiving IVIg. Samples and clinical data were obtained prior to IVIg/SCIg and at 3 days, 7 days, and, in CIDP patients receiving IVIg, 21 days post-administration. Serum cytokines were assessed by Luminex-based multiplex assay and enzyme-linked immunosorbent assay. Immune cells were characterized by flow cytometry. RESULTS: Immune cell profiles of CIDP and CVID patients differed in frequencies of myeloid dendritic cells and cytotoxic natural killer cells. During treatment with IVIg or SCIg in CIDP patients, cellular immunomarkers were largely similar. CIDP patients receiving IVIg had higher macrophage inflammatory protein (MIP)-1α (p = 0.01), interleukin (IL)-4 (p = 0.04), and IL-33 (p = 0.04) levels than SCIg recipients. IVIg treatment more broadly modulated cytokines in CIDP than SCIg treatment. CONCLUSIONS: Our study demonstrates that the modulation of cellular immunomarkers in CIDP is independent of the application route of therapeutic immunoglobulin. Minor differences were observed between CIDP and CVID patients. In contrast, cytokines were differentially modulated by IVIg and SCIg in CIDP.

Kinesin-5 inhibition improves neural regeneration in experimental autoimmune neuritis.

BACKGROUND: Autoimmune neuropathies can result in long-term disability and incomplete recovery, despite adequate first-line therapy. Kinesin-5 inhibition was shown to accelerate neurite outgrowth in different preclinical studies. Here, we evaluated the potential neuro-regenerative effects of the small molecule kinesin-5 inhibitor monastrol in a rodent model of acute autoimmune neuropathies, experimental autoimmune neuritis. METHODS: Experimental autoimmune neuritis was induced in Lewis rats with the neurogenic P2-peptide. At the beginning of the recovery phase at day 18, the animals were treated with 1 mg/kg monastrol or sham and observed until day 30 post-immunisation. Electrophysiological and histological analysis for markers of inflammation and remyelination of the sciatic nerve were performed. Neuromuscular junctions of the tibialis anterior muscles were analysed for reinnervation. We further treated human induced pluripotent stem cells-derived secondary motor neurons with monastrol in different concentrations and performed a neurite outgrowth assay. RESULTS: Treatment with monastrol enhanced functional and histological recovery in experimental autoimmune neuritis. Motor nerve conduction velocity at day 30 in the treated animals was comparable to pre-neuritis values. Monastrol-treated animals showed partially reinnervated or intact neuromuscular junctions. A significant and dose-dependent accelerated neurite outgrowth was observed after kinesin-5 inhibition as a possible mode of action. CONCLUSION: Pharmacological kinesin-5 inhibition improves the functional outcome in experimental autoimmune neuritis through accelerated motor neurite outgrowth and histological recovery. This approach could be of interest to improve the outcome of autoimmune neuropathy patients.

Subclinical motor involvement in nonsystemic vasculitic neuropathy determined by the motor unit number estimation method MScanFit.

INTRODUCTION/AIMS: Nonsystemic vasculitic neuropathy (NSVN) is characterized by a predominant lower limb involvement in many patients. Motor unit changes in upper extremity muscles have not been investigated in this subgroup but may be of interest for improving our understanding of the multifocal nature of the disease and counseling of patients about potential future symptoms. In this study we aimed to better understand subclinical motor involvement in the upper extremity muscles of patients with lower limb-predominant NSVN using the new motor unit number estimation (MUNE) method MScanFit. METHODS: In this single-center, cross-sectional study, 14 patients with biopsy-proven NSVN, with no clinical signs of upper extremity motor involvement, were investigated and compared with 14 age-matched healthy controls. All participants were assessed clinically and by the MUNE method MScanFit to the abductor pollicis brevis muscle. RESULTS: The number of motor units and peak CMAP amplitudes were significantly reduced in patients with NSVN (P = .003 and P = .004, respectively). Absolute median motor unit amplitudes and CMAP discontinuities were not significantly different (P = .246 and P = .1, respectively). CMAP discontinuities were not significantly correlated with motor unit loss (P = .15, rho = 0.4). The number of motor units did not correlate with clinical scores (P = .77, rho = 0.082). DISCUSSION: Both MUNE and CMAP amplitudes showed motor involvement in upper extremity muscles in lower limb-predominant NSVN. Overall, there was no evidence of significant reinnervation. Investigations of the abductor pollicis brevis muscle did not show a correlation with overall functional disability of the patients.

The gut microbiome in intravenous immunoglobulin-treated chronic inflammatory demyelinating polyneuropathy.

BACKGROUND AND PURPOSE: The gut microbiome is involved in autoimmunity. Data on its composition in chronic inflammatory demyelinating polyneuropathy (CIDP), the most common chronic autoimmune disorder of peripheral nerves, are currently lacking. METHODS: In this monocentric exploratory pilot study, stool samples were prospectively collected from 16 CIDP patients (mean age 58 ± 10 years, 25% female) before and 1 week after administration of intravenous immunoglobulin (IVIg). Gut microbiota were analyzed via bacterial 16S rRNA gene sequencing and compared to 15 age-matched healthy subjects (mean age 59 ± 15 years, 66% female). RESULTS: The gut microbiota of CIDP patients showed an increased alpha-diversity (p = 0.005) and enrichment of Firmicutes, such as Blautia (p = 0.0004), Eubacterium hallii (p = 0.0004), or Ruminococcus torques (p = 0.03), and of Actinobacteriota (p = 0.03) compared to healthy subjects. IVIg administration did not alter the gut microbiome composition in CIDP in this short-term observation (p = 0.95). CONCLUSIONS: The gut microbiome in IVIg-treated CIDP shows distinct features, with increased bacterial diversity and enrichment of short-chain fatty acid producing Firmicutes. IVIg had no short-term impact on the gut microbiome in CIDP patients. As the main limitation of this exploratory pilot study was small cohort size, future studies also including therapy-naïve patients are warranted to verify our findings and to explore the impact of long-term IVIg treatment on the gut microbiome in CIDP.

Antibody response after COVID-19 vaccination in intravenous immunoglobulin-treated immune neuropathies.

BACKGROUND AND PURPOSE: This study assessed the prevalence of anti-SARS-CoV-2 antibodies in therapeutic immunoglobulin and their impact on serological response to COVID-19 mRNA vaccine in patients with intravenous immunoglobulin (IVIg)-treated chronic immune neuropathies. METHODS: Forty-six samples of different brands or lots of IVIg or subcutaneous IgG were analyzed for anti-SARS-CoV-2 IgG using enzyme-linked immunosorbent assay and chemiluminescent microparticle immunoassay. Blood sera from 16 patients with immune neuropathies were prospectively analyzed for anti-SARS-CoV-2 IgA, IgG, and IgM before and 1 week after IVIg infusion subsequent to consecutive COVID-19 mRNA vaccine doses and after 12 weeks. These were compared to 42 healthy subjects. RESULTS: Twenty-four (52%) therapeutic immunoglobulin samples contained anti-SARS-CoV-2 IgG. All patients with immune neuropathies (mean age = 65 ± 16 years, 25% female) were positive for anti-SARS-CoV-2 IgG after COVID-19 vaccination. Anti-SARS-CoV-2 IgA titers significantly decreased 12-14 weeks after vaccination (p = 0.02), whereas IgG titers remained stable (p = 0.2). IVIg did not significantly reduce intraindividual anti-SARS-CoV-2 IgA/IgG serum titers in immune neuropathies (p = 0.69). IVIg-derived anti-SARS-CoV-2 IgG did not alter serum anti-SARS-CoV-2 IgG decrease after IVIg administration (p = 0.67). CONCLUSIONS: Our study indicates that IVIg does not impair the antibody response to COVID-19 mRNA vaccine in a short-term observation, when administered a minimum of 2 weeks after each vaccine dose. The infusion of current IVIg preparations that contain anti-SARS-CoV-2 IgG does not significantly alter serum anti-SARS-CoV-2 IgG titers.

Emerging treatment landscape for Guillain-Barré Syndrome (GBS): what's new?

INTRODUCTION: Guillain-Barré syndrome (GBS) is a monophasic immune neuropathic disorder characterized by acute paralysis. A significant portion of patients are left with residual deficits, which presents a considerable global healthcare challenge. The precise mechanisms underlying GBS pathogenesis are not fully elucidated. Recent studies have focused on postinfectious molecular mimicry and identified involvement of IgG autoantibodies and innate immune effectors in GBS. Intravenous immunoglobulins (IVIg) and plasma exchange (PE) are two established evidence-based immunomodulatory treatments for GBS, but a significant proportion of GBS patients fails to respond adequately to either therapy. This emphasizes an urgent need for novel and more potent treatments. AREAS COVERED: We discuss novel immunomodulatory therapies presently at different phases of preclinical and clinical investigation. Some drugs in development target pathophysiologic mechanisms such as IgG autoantibody catabolism and complement activation, which are relevant to GBS. EXPERT OPINION: There is an unmet need for more effective immune therapies for GBS. New immunomodulatory therapies under development may provide more potent options for GBS patients who do not respond to IVIg or PE. Future directions may include incorporating neuroprotective interventions based on evolving understanding of mechanisms underlying nerve injury and axonal degeneration.

Rejuvenating fecal microbiota transplant enhances peripheral nerve repair in aged mice by modulating endoneurial inflammation.

Peripheral nerve injury (PNI) resulting from trauma or neuropathies can cause significant disability, and its prognosis deteriorates with age. Emerging evidence suggests that gut dysbiosis and reduced fecal short-chain fatty acids (SCFAs) contribute to an age-related systemic hyperinflammation (inflammaging), which hinders nerve recovery after injury. This study thus aimed to evaluate the pro-regenerative effects of a rejuvenating fecal microbiota transplant (FMT) in a preclinical PNI model using aged mice. Aged C57BL/6 mice underwent bilateral crush injuries to their sciatic nerves. Subsequently, they either received FMT from young donors at three and four days after the injury or retained their aged gut microbiota. We analyzed gut microbiome composition and SCFA concentrations in fecal samples. The integrity of the ileac mucosal barrier was assessed by immunofluorescence staining of Claudin-1. Flow cytometry was utilized to examine immune cells and cytokine production in the ileum, spleen, and sciatic nerve. Various assessments, including behavioural tests, electrophysiological studies, and morphometrical analyses, were conducted to evaluate peripheral nerve function and repair following injury. Rejuvenating FMT reversed age-related gut dysbiosis by increasing Actinobacteria, especially Bifidobacteriales genera. This intervention also led to an elevation of gut SCFA levels and mitigated age-related ileac mucosal leakiness in aged recipients. Additionally, it augmented the number of T-helper 2 (Th2) and regulatory T (Treg) cells in the ileum and spleen, with the majority being positive for anti-inflammatory interleukin-10 (IL-10). In sciatic nerves, rejuvenating FMT resulted in increased M2 macrophage counts and a higher IL-10 production by IL-10+TNF-α- M2 macrophage subsets. Ultimately, restoring a youthful gut microbiome in aged mice led to improved nerve repair and enhanced functional recovery after PNI. Considering that FMT is already a clinically available technique, exploring novel translational strategies targeting the gut microbiome to enhance nerve repair in the elderly seems promising and warrants further evaluation.

Antibiotics-Induced Intestinal Immunomodulation Attenuates Experimental Autoimmune Neuritis (EAN).

BACKGROUND: The composition of gut microbiota plays a pivotal role in priming the immune system and thus impacts autoimmune diseases. Data on the effects of gut bacteria eradication via systemic antibiotics on immune neuropathies are currently lacking. This study therefore assessed the effects of antibiotics-induced gut microbiota alterations on the severity of experimental autoimmune neuritis (EAN), a rat model of Guillain-Barré Syndrome (GBS). Myelin P0 peptide 180-199 (P0 180-199)-induced EAN severity was compared between adult Lewis rats (12 weeks old) that received drinking water with or without antibiotics (colistin, metronidazole, vancomycin) and healthy rats, beginning antibiotics treatment immediately after immunization (day 0), and continuing treatment for 14 consecutive days. Neuropathy severity was assessed via a modified clinical score, and then related to gut microbiota alterations observed after fecal 16S rRNA gene sequencing at baseline and after EAN induction. Effectors of gut mucosal and endoneurial immunity were assessed via immunostaining. EAN rats showed increased gut mucosal permeability alongside increased mucosal CD8+ T cells compared to healthy controls. Antibiotics treatment alleviated clinical EAN severity and reduced endoneurial T cell infiltration, decreased gut mucosal CD8+ T cells and increased gut bacteria that may be associated with anti-inflammatory mechanisms, like Lactobacillus or Parasutterella. Our findings point out a relation between gut mucosal immunity and the pathogenesis of EAN, and indicate that antibiotics-induced intestinal immunomodulation might be a therapeutic approach to alleviate autoimmunity in immune neuropathies. Further studies are warranted to evaluate the clinical transferability of these findings to patients with GBS.

Motor unit number estimation by MScanFit in myotonic dystrophies.

BACKGROUND: MScanFit is a new motor unit number estimation (MUNE) technique applied in motor neuron diseases and polyneuropathies to assess clinical progression and underlying disease pathology. So far, its value in myopathies, especially myotonic dystrophies (MD), has not been investigated. METHODS: Motor unit loss and characteristics of patients with genetically confirmed MD type 1 (n = 7) and type 2 (n = 5) were investigated using MScanFit of the abductor pollicis brevis muscle and compared to age-matched healthy controls. MUNE measures were correlated with muscle impairment determined by the MRC sum score and handgrip strength. RESULTS: MScanFit detected motor unit loss in patients with MD (p = 0.017). There was no significant difference in motor unit loss between MD type 1 and type 2 (p = 0.64). CMAP-discontinuities which, when added up, exceed 50% of the CMAP amplitude were reduced in MD patients (p = 0.0284), but motor unit amplitudes were not significantly different (p = 0.0597). The motor unit loss strongly correlated with the MRC sum score (p = 0.014, Rho = 0.678). CONCLUSIONS: Our study shows the feasibility of MScanFit in MD and its potential to serve as a surrogate marker for overall muscle impairment. Motor unit analysis indicates that neurogenic alterations in both MD subtypes might be present.

CIDP: Analysis of Immunomarkers During COVID-19 mRNA-Vaccination and IVIg-Immunomodulation: An Exploratory Study.

Availability of COVID-19 mRNA vaccine for patients with chronic inflammatory demyelinating polyneuropathy (CIDP) treated with intravenous immunoglobulin (IVIg) raises the question of whether COVID-19 mRNA vaccine influences disease activity or IVIg-mediated immunomodulation in CIDP. In this exploratory study, blood samples of CIDP patients on IVIg treatment were longitudinally analyzed before and after vaccination with a COVID-19 mRNA vaccine. A total of 44 samples of eleven patients were characterized at four timepoints by ELISA and flow cytometry in terms of immunomarkers for disease activity and IVIg-immunomodulation. Apart from a significantly lower expression of CD32b on naïve B cells after vaccination, no significant alteration of immunomarkers for CIDP or IVIg-mediated immunomodulation was observed. Our exploratory study suggests that COVID-19 mRNA vaccine does not have a relevant impact on immune activity in CIDP. In addition, immunomodulatory effects of IVIg in CIDP are not altered by COVID-19 mRNA vaccine. This study was registered in the German clinical trial register (DRKS00025759). Overview over the study design. Blood samples of CIDP patients on recurrent IVIg treatment and vaccination with a COVID-19 mRNA vaccine were obtained at four timepoints for cytokine ELISA and flow cytometry, to assess key cytokines and cellular immunomarkers for disease activity and IVIg-immunomodulation in CIDP.

2,4-Dinitrophenol does not exert neuro-regenerative potential in experimental autoimmune neuritis.

OBJECTIVE: We evaluated the potential neuro-regenerative effects of the mitochondrial uncoupler 2,4-Dinitrophenol in experimental autoimmune neuritis, an animal model for an acute autoimmune neuropathy. METHODS: Experimental autoimmune neuritis was induced in Lewis rats. Different concentrations of 2,4-Dinitrophenol (1 mg/kg, 0.1 mg/kg and 0.01 mg/kg) were applied during the recovery phase of the neuritis (at days 18, 22 and 26) and compared to the vehicle. Any effects were assessed through functional, electrophysiological, and morphological analysis via electron microscopy of all groups at day 30. Additional immune-histochemical analysis of inflammation markers and remyelination of the sciatic nerves were performed for the dosage of 1 mg/kg and control. RESULTS: No enhancement of functional or electrophysiological recovery was observed in all 2,4-Dinitrophenol-treated groups. Cellular inflammation markers of T cells (CD3+) were comparable to control, and an increase of macrophages (IbA1+) invasion in the sciatic nerves was observed. Treatment with 2,4-Dinitrophenol reduced axonal swelling in myelinated and unmyelinated fibers with an increased production of brain-derived neurotrophic factor. CONCLUSION: Our findings do not support the hypothesis that repurposing of the mitochondrial uncoupler 2,4-Dinitrophenol exerts functionally relevant neuro-regenerative effects in autoimmune neuritis.

Chronic Inflammatory Demyelinating Polyneuropathy (CIDP): Current Therapies and Future Approaches.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired immune-mediated polyradiculoneuropathy leading to disability via inflammatory demyelination of peripheral nerves. Various therapeutic approaches with different mechanisms of action are established for the treatment of CIDP. Of those, corticosteroids, intravenous or subcutaneous immunoglobulin, or plasma exchange are established first-line therapies as suggested by the recently revised EAN/PNS guidelines for the management of CIDP. In special cases, immunosuppressants or rituximab may be used. Novel therapeutic approaches currently undergoing clinical studies include molecules or monoclonal antibodies interacting with Fc receptors on immune cells to alleviate immune-mediated neuronal damage. Despite various established therapies and the current development of novel therapeutics, treatment of CIDP remains challenging due to an heterogeneous disease course and the lack of surrogate parameters to predict the risk of clinical deterioration. This review summarizes established therapies for CIDP and provides an outlook on future therapeutic approaches.

Amyloidogenicity assessment of transthyretin gene variants.

OBJECTIVE: Hereditary transthyretin-mediated amyloidosis is a treatable condition caused by amyloidogenic variants in the transthyretin-gene resulting in severe peripheral neuropathy or cardiomyopathy. Only about a third of over 130 known variants are clearly pathogenic, most are classified as variants of uncertain significance. A clear delineation of these into pathogenic or non-pathogenic is highly desirable but hampered by low frequency and penetrance. We thus sought to characterize their amylogenic potential by an unbiased in vitro approach. METHODS: Thioflavin T and turbidity assays were used to compare the potential of mammalian cell expressed wt-transthyretin and 12 variant proteins (either variants of uncertain significance, benign, pathogenic) to aggregate and produce amyloid fibrils in vitro. As proof of principle, the assays were applied to transthyretin-Ala65Val, a variant that was newly detected in a family with peripheral neuropathy and amyloid deposits in biopsies. In silico analysis was performed to compare the position of the benign and pathogenic variants. RESULTS: Transthyretin-Ala65Val showed a significantly higher amyloidogenic potential than wt-transthyretin, in both turbidity- and Thioflavin T-assays, comparable to known pathogenic variants. The other eight tested variants did not show an increased amyloidogenic potential. In silico structural analysis further confirmed differences between pathogenic and benign variants in position and interactions. INTERPRETATION: We propose a biochemical approach to assess amyloidogenic potential of transthyretin variants. As exemplified by transthyretin-Ala65Val, data of three assays together with histopathology clearly demonstrates its amyloidogenicity.

Clinical management of chronic inflammatory demyelinating polyneuropathy (CIDP) in Europe and India: An exploratory study.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an autoimmune disorder causing inflammatory demyelination of peripheral nerves and consecutive disability. Diagnostic criteria and treatments are well established, but it is unknown how clinical practice may differ in different geographical regions. In this multicentre study, clinical management of CIDP was compared in 44 patients from Germany, India and Norway regarding diagnostic and therapeutic procedures. All centres used EFNS/PNS diagnostic criteria for CIDP but diagnostic workup varied regarding screening for infectious diseases, genetic testing and nerve biopsy. Intravenous immunoglobulin and prednisolone were the most common therapies in all centres with differences in indication and dosage. Patients from the Indian cohort were the most severely affected with less diverse therapeutic approaches, whereas psychological strain did not differ significantly from the two other cohorts. Our exploratory study discloses an unaddressed issue in management of CIDP that should be further investigated to optimise standard of care for CIDP worldwide.

Changes of Serum IgG Dimer Levels after Treatment with IVIg in Guillain-Barré Syndrome.

Intravenous immunoglobulins (IVIg) are standard treatment for Guillain-Barré syndrome (GBS). Their exact mechanisms of action are versatile and not fully understood. One possible mechanism is neutralization of circulating autoantibodies via binding to anti- idiotypic antibodies forming idiotype-anti-idiotype dimeric IgG immune complexes. To examine the role of immune complex formation as mechanism of action for IVIg in GBS, 34 C57Bl/6 mice were either treated with anti-ganglioside antibodies and IVIg or IVIg and PBS alone, whereas eight additional mice were treated either with anti-ganglioside autoantibodies and IVIg or anti-ganglioside autoantibodies alone. Subsequently IgG dimer formation was assessed by high performance liquid chromatography (HPLC) and enzyme- linked immunosorbent assay (ELISA). In addition, IgG dimer formation was measured in sera of eight GBS patients who were treated with IVIg. In mice, a significant increase of dimeric IgG after administration of anti-ganglioside antibodies and IVIg could be observed. Re-monomerized IgG dimers showed immunoreactivity against gangliosides and serum immunoreactivity was significantly reduced after IVIg infusion. Likewise also in GBS patients, IgG dimer formation could be detected after IVIg treatment. Our data indicate that dimeric IgG immune complexes contain anti-idiotypic antibodies and provide proof of concept that IVIg treatment in GBS results in measurable amounts of IgG dimers. Larger patient cohorts are needed to evaluate serum IgG dimer increase as a possible marker for treatment response in GBS. Graphical Abstract Mechanism of action: Intravenous immunoglobulins (IVIg) and anti-ganglioside antibodies form dimeric IgG immune complexes, preventing axonal damage in Guillain-Barré Syndrome.

Impact of Age and Polytherapy on Fingolimod Induced Bradycardia: a Preclinical Study.

Fingolimod is a an oral disease modifying drug for relapsing remitting multiple sclerosis (MS) preventing egress of B and T cells from lymph nodes. Relevant first dose adverse events include bradycardia and atrioventricular conduction slowing. Cardiac side effects of fingolimod and combinational pharmacotherapy including duloxetine and tolterodine were monitored in mice of different age using implantable ECG telemetric systems. Cardiac tissue was assessed for S1P-receptor subtype (1 and 3), and for GIRK1 expression. Fingolimod led to a significant heart rate reduction within 60 min, which returned to baseline values within 24 h. In older mice bradycardia was more pronounced compared to younger mice. Atrioventricular conduction was not affected. Older mice showed a higher S1PR3 expression in a naïve state and receptor expression was reduced after fingolimod administration. Combination with duloxetine or tolterodine alleviated fingolimod induced heart rate decrease. Our data provide preclinical evidence that negative chronotropic effects of fingolimod might be age dependent, possibly due to an altered expression and internalization of cardiac S1PR3 in older animals. This data could be relevant for future clinical monitoring and patient selection in the aging MS population. Combinational therapies of fingolimod and duloxetine or tolterodine are well tolerated and safe without an increased risk for pronounced bradycardia or arrhythmia.