Rindera graeca (A. DC.) Boiss. & Heldr. (Boraginaceae) In Vitro Cultures Targeting Lithospermic Acid B and Rosmarinic Acid Production.

The in vitro cultures of Rindera graeca, a rare endemic plant, were developed as a sustainable source of phenolic acids. Various shoot and root cultures were established and scaled up in a sprinkle bioreactor. A multiplication rate of 7.2 shoots per explant was achieved. HPLC-PDA-ESI-HRMS analysis revealed the presence of rosmarinic acid (RA) and lithospermic acid B (LAB) as the main secondary metabolites in both the shoot and root cultures. The maximum RA (30.0 ± 3.2 mg/g DW) and LAB (49.3 ± 15.5 mg/g DW) yields were determined in root-regenerated shoots. The strongest free radical scavenging activity (87.4 ± 1.1%), according to 2,2-diphenyl-1-picrylhydrazyl-hydrate assay, was noted for roots cultivated in a DCR medium. The highest reducing power (2.3 µM ± 0.4 TE/g DW), determined by the ferric-reducing antioxidant power assay, was noted for shoots cultivated on an SH medium containing 0.5 mg/L 6-benzylaminopurine. A genetic analysis performed using random amplified polymorphic DNA and start codon targeted markers revealed genetic variation of 62.8% to 96.5% among the investigated shoots and roots. This variability reflects the capacity of cultivated shoots and roots to produce phenolic compounds.

Value of short exercise and short exercise with cooling tests in diagnosis of recessive form of myotonia congenita (Becker disease) - are sex differences important?

INTRODUCTION: In myotonia congenita (MC), activation with exercise or cooling can induce transient changes in compound motor action potential (CMAP) parameters, thus providing a guide to genetic analysis. MATERIAL AND METHODS: We performed the short exercise test (SET) and the short exercise test with cooling (SETC) in 30 patients with genetically confirmed Becker disease (BMC) to estimate their utility in the diagnosis of BMC. RESULTS: Although we observed a significant decrease in CMAP amplitude immediately after maximal voluntary effort in both tests in the whole BMC group, in men this decline was significantly smaller than in women, especially in SET. Clinical implications/future directions: In men with a clinical suspicion of BMC, a small decrease in CMAP amplitude in SET together with a typical decline in SETC does not exclude the diagnosis of BMC. Our results show a sex-specific difference in chloride channel function in BMC, which needs further investigation.

Motor neurons loss in Parkinson Disease: An electrophysiological study (MUNE).

The aim of this study was to evaluate the involvement of a peripheral motor neuron in Parkinson Disease (PD) using the motor unit number estimation (MUNE) method, which reflects motor unit loss in motor neuron diseases. Multipoint incremental MUNE method was calculated in abductor pollicis brevis (APB) and abductor digiti minimi (ADM) in forty one (41) patients with PD and forty five (45) healthy volunteers. From the analysis, the MUNE of APB was lower in PD than in the control group, especially in the sub-group aged 60 years or older. MUNE was negatively correlated with the age of patientsfor APB, but not with the duration of the disease and advancement of PD. The loss of motor units in sporadic Parkinson's disease revealed by multipoint incremental MUNE method is considered a sign of lower motor neuron involvement, however, loss of motor neurons is slight and does not manifest equally in all muscles . Thus, the results from this experiment should be treated with concern, as it could be a landmark for further experiments.

Migraine and Its Association with Hyperactivity of Cell Membranes in the Course of Latent Magnesium Deficiency-Preliminary Study of the Importance of the Latent Tetany Presence in the Migraine Pathogenesis.

So far, there is no consistent and convincing theory explaining the pathogenesis of migraines. Vascular disorders, the effect of oxidative stress on neurons, and the contribution of magnesium-calcium deficiencies in triggering cortical depression and abnormal glutaminergic neurotransmission are taken into account. However, there are no reliable publications confirming the role of dietary deficits of magnesium and latent tetany as factors triggering migraine attacks. The aim of the study was to evaluate the influence of latent magnesium deficiency assessed with the electrophysiological tetany test on the course of migraine. The study included: a group of 35 patients (29 women and six men; in mean age 41 years) with migraine and a control group of 24 (17 women and seven men; in mean age 39 years) healthy volunteers. Migraine diagnosis was based on the International Headache Society criteria, 3rd edition. All patients and controls after full general and neurological examination were subjected to a standard electrophysiological ischemic tetany test. Moreover, the level of magnesium in blood serum was tested and was in the normal range in all patients. Then, the incidence of a positive tetany EMG test results in the migraine group and the results in the subgroups with and without aura were compared to the results in the control group. Moreover, the relationship between clinical markers of spasmophilia and the results of the tetany test was investigated in the migraine group. As well as the relationship between migraine frequency and tetany test results. There was no statistically significant difference in the occurrence of the electrophysiological exponent of spasmophilia between the migraine and control group. Neither correlation between the occurrence of clinical symptoms nor the frequency of migraine attacks and the results of the tetany test was stated (p > 0.05). However, there was an apparent statistical difference between the subgroup of migraine patients with aura in relation to the control group (p < 0.05). The result raises hope to find a trigger for migraine attacks of this clinical form, the more that this factor may turn out to be easy to supplement with dietary supplementation.

Differentiation between single fiber potentials from one muscle fiber or contaminated by other fibers using discriminating function.

OBJECTIVES: The aim was to improve the identification of potentials recorded using single fiber electromyography (SFEMG) contaminated by potentials from other muscle fibers, which might affect measured jitter value, by defining more selective criteria of single fiber potential (SFP) discrimination. We were looking for solutions suitable for automatization. METHODS: Standard parameters characterizing SFP and their combinations were analyzed to define an analytical discriminating function able to verify if potentials recorded using SFEMG are due to single fiber or due to two (or more) fibers. RESULTS: The discriminating function is based on combination of standard SFP parameters. The procedure was tested on a set of simulated i.e., known data and on samples of clinical data. The tests on simulated data confirmed assumed properties of discriminating function. Preliminary results of pilot studies using patient data suggest its ability for differentiation between potentials of one fiber and contaminated ones. The procedure is suitable for automatization. CONCLUSION: Results suggest that proposed discriminating function when supplementing standard criteria would help to promote SFP recordings and enable to improve relevancy of jitter measurements and of jitter value norms.

The needle EMG findings in myotonia congenita.

INTRODUCTION: Myotonia congenita (MC) is caused by pathogenic variants in the CLCN1 gene coding the chloride channel protein. METHODS: To test the hypothesis that needle EMG could be helpful in distinguishing between the recessive and dominant MC, we performed EMG examination in 36 patients (23 men) aged 4-61 years with genetically proven MC: in 30 patients with autosomal recessive MC (Becker MC) and in 6 with autosomal dominant MC (Thomsen MC). RESULTS: Myotonic discharges were recorded in 95.8% of examined muscles. For the whole MC group we observed a significant positive correlation between parameters of motor unit activity potentials (MUAPs) in vastus lateralis and tibialis anterior muscles and the duration of the disease. Similar correlation for biceps brachii also was found in Becker MC subgroup only. DISCUSSION: EMG could still be helpful in diagnosis of MC and together with provocative tests might be useful in differentiation between recessive and autosomal MC.

Electrodiagnostics: MUNE and MUNIX as methods of estimating the number of motor units - biomarkers in lower motor neurone disease.

Routine quantitative electromyography is used for the assessment of the presence of lower motor neurone involvement and its consequences, including primary denervation and compensatory reinnervation of muscle fibres. However, it is not useful for the assessment of the motor unit number reserve. The need for a valid biomarker to evaluate lower motor neurone disease progression in such diseases as amyotrophic lateral sclerosis, and for use in clinical trials, has led to a number of studies of the methods that allow assessment of the number of motor units. In this review, motor unit number estimation (MUNE) methods with incremental stimulation and the recently developed motor unit number index (MUNIX) method, along with their technical and clinical aspects, are presented as methods which reflect motor unit loss in neurogenic processes. These electrodiagnostic tests may allow a valuable assessment of disease progression and the efficacy of new therapeutic methods in clinical trials in diseases with lower motor neurone degeneration.

Assessment of the reliability of the motor unit size index (MUSIX) in single subject "round-robin" and multi-centre settings.

OBJECTIVE: The motor unit size index (MUSIX) is incorporated into the motor unit number index (MUNIX). Our objective was to assess the intra-/inter-rater reliability of MUSIX in healthy volunteers across single subject "round robin" and multi-centre settings. METHODS: Data were obtained from (i) a round-robin assessment in which 12 raters (6 with prior experience and 6 without) assessed six muscles (abductor pollicis brevis, abductor digiti minimi, biceps brachii, tibialis anterior, extensor digitorum brevis and abductor hallucis) and (ii) a multi-centre study with 6 centres studying the same muscles in 66 healthy volunteers. Intra/inter-rater data were provided by 5 centres, 1 centre provided only intra-rater data. Intra/inter-rater variability was assessed using the coefficient of variation (COV), Bland-Altman plots, bias and 95% limits of agreement. RESULTS: In the round-robin assessment intra-rater COVs for MUSIX ranged from 7.8% to 28.4%. Inter-rater variability was between 7.8% and 16.2%. Prior experience did not impact on MUSIX values. In the multi-centre study MUSIX was more consistent than the MUNIX. Abductor hallucis was the least reliable muscle. CONCLUSIONS: The MUSIX is a reliable neurophysiological biomarker of reinnervation. SIGNIFICANCE: MUSIX could provide insights into the pathophysiology of a range of neuromuscular disorders, providing a quantitative biomarker of reinnervation.

Motor Unit Number Index (MUNIX) as a biomarker of motor unit loss in post-polio syndrome versus needle EMG.

MUNIX method (Motor Unit Number Index) had been not used to assess number of motor neurons in post-polio syndrome in contrary to needle electromyography. OBJECTIVES: To confirm if MUNIX reflects motor unit loss and clinical stage and to assess difference in MUNIX and EMG results between muscles in different stage. METHODS: 132 Muscles (MUNIX) and 96 (EMG) in 12 patients were studied and divided into groups: with normal strength(N), stable weakness and atrophy(S), new weakness and atrophy(W). RESULTS: In PPS group MUNIX global was 561.36 ±â€¯282.6 (right 6 muscles) and 561.27 ±â€¯281.1 (left) significantly lower than in control group (six muscles 1139.6 ±â€¯164.5) (p < 0.05). MUNIX global correlated with MRC global. MUNIX was greater in muscles with normal strength (95-100% of normal values) than in those with stable weakness (48%-0% of normal values) and new weakness (65%-0% of normal values). Respectively to clinical stage of muscle MUP (motor unit potential) amplitude increased to 350% of normal value, from 250% to 110%, and from 300% to 700%. No correlation was found between MUP parameters and MRC values. CONCLUSIONS: MUNIX reflects motor dysfunction and could be a good biomarker for loss of motor neurons in PPS.

Identification of components from distant fibers in a recorded single muscle fiber potential (SFP) - a new approach to the SFP criteria.

OBJECTIVES: The aim of our study was to verify the effectiveness of single fiber potential (SFP) criteria in cases when the potential recorded using single fiber electrode (SFE) or concentric needle electrode (CNE) is contaminated by distant fibers. METHODS: Morphological counterparts of SFP were studied using computer simulations. In this study, we examined triphasic potentials using a model of a linear source of SFP. The criteria defining SFP in the case of SFP contaminated by distant fibers were analyzed, and the effect of second fiber contamination on jitter and fiber diameter determination evaluated. RESULTS: We found that SFP criteria prevent detection of SFP from fibers smaller than about 30µm in diameter, but do not prevent classification of a potential as an SFP even though it is formed by two or more fibers. This suggests that the presently used criteria may lead to incorrect interpretation of SFP potentials. SFPs contaminated by fibers of diameters differing by a few percent fulfill the criteria but a negative peak may be shifted in time and therefore impact jitter and diameter measurements. This contamination generally tends to decrease both the jitter and the determined diameter. A new approach to the identification of SFP is presented, determining fiber diameter and distance from the electrode to enable maximum sensitivity to potential contamination by the effect of a second fiber. CONCLUSION: A new parameter characterizing SFP shape changes is introduced. This parameter is used in the method by which additional fibers affecting the SFP may be detected.

Hypoglossal nerve palsy as an isolated syndrome of internal carotid artery dissection: A review of the literature and a case report.

A review of literature on the dissection of internal carotid artery was presented with a presentation of a rare case of patient with transient left hypoglossal nerve palsy caused by mechanic compression from intramural hematoma in higher extracranial portion of dissected carotid artery confirmed in MRI and CT scans. The clinical presentation and management are discussed.

Intraspinal Transplantation of the Adipose Tissue-Derived Regenerative Cells in Amyotrophic Lateral Sclerosis in Accordance with the Current Experts' Recommendations: Choosing Optimal Monitoring Tools.

Stem cells (SCs) may constitute a perspective alternative to pharmacological treatment in neurodegenerative diseases. Although the safety of SC transplantation has been widely shown, their clinical efficiency in amyotrophic lateral sclerosis (ALS) is still to be proved. It is not only due to a limited number of studies, small treatment groups, and fast but nonlinear disease progression but also due to lack of objective methods able to show subtle clinical changes. Preliminary guidelines for cell therapy have recently been proposed by a group of ALS experts. They combine clinical, neurophysiological, and functional assessment together with monitoring of the cytokine level. Here, we describe a pilot study on transplantation of autologous adipose-derived regenerative cells (ADRC) into the spinal cord of the patients with ALS and monitoring of the results in accordance with the current recommendations. To show early and/or subtle changes within the muscles of interest, a wide range of clinical and functional tests were used and compared in order to choose the most sensitive and optimal set. Additionally, an analysis of transplanted ADRC was provided to develop standards ensuring the derivation and verification of adequate quality of transplanted cells and to correlate ADRC properties with clinical outcome.

Comprehensive evaluation of EMG and biopsy findings supported by computer simulations - A preliminary study.

OBJECTIVE: The aim was to compare muscle fiber diameters obtained from standard muscle biopsy and from computer simulations based on recorded motor unit potentials (MUPs). METHODS: Electromyography (EMG) and muscle biopsy were performed in 14 patients with a suspicion of a neuromuscular disorder. Histograms of the simulated muscle fiber diameters (SMFDs) were compared with those from the biopsy RESULTS: The values of the SMFDs were similar to those in the muscle biopsy for the same patient (p = 0.05) in all 14 cases. CONCLUSIONS: Comprehensive evaluation of EMG and biopsy findings supported by computer simulations may help resolve the discrepancy between the assessment of muscle by EMG and biopsy by explaining different results obtained with these two methods. SIGNIFICANCE: Evaluation of the SMFDs that are comparable to biopsy findings extends the amount of information available from EMG.

Electromyographic findings in sporadic inclusion body myositis.

INTRODUCTION: Clinically oriented diagnostic criteria can be as specific for diagnosis of sporadic inclusion body myositis (sIBM) as pathological criteria, especially at the time of presentation. EMG may provide an convincing proof that a muscle biopsy should be performed. AIMS: To compare the EMG results in patients with sIBM divided into subgroups based on the newest ENMC criteria for sIBM and to obtain the utility of EMG in the diagnostic process at the time of presentation. METHODS: We retrospectively analysed 16 patients with sIBM for motor unit action potential (MUAP) morphology as well as occurrence and distribution of abnormal spontaneous activity (SA) in muscles. RESULTS: Abnormal SA was recorded in 62.5% of sIBM patients. We found statistically significant differences between subgroups in the incidence of polyphasic MUAPs and high amplitude outliers which were more commonly seen in the "clinico-pathologically defined sIBM". Duration of MUAP in the tibialis anterior was significantly shorter in "probable sIBM". DISCUSSION: "Pseudo-neurogenic" MUAPs, mainly in lower limb muscles, are more commonly seen in "clinico-pathologically defined sIBM" while myopathic MUAPs with prominent abnormal SA are recorded in patients diagnosed with "probable sIBM". Both EMG patterns may be suggestive of sIBM and be an indication for further diagnosis.

Abnormal spontaneous activity in primary myopathic disorders.

INTRODUCTION: Reproducible non-insertional spontaneous activity (SA), with the exception of endplate activity, is an unequivocal sign of abnormality and is one of the most useful findings obtained on electromyography. METHODS: In this retrospective study we analyzed occurrence and distribution of abnormal SA in 151 patients with genetically confirmed myopathies. RESULTS: Complex repetitive discharges (CRDs) occurred more frequently than fibrillation potentials (fibs) and positive sharp waves (PSWs) in centronuclear myopathy (CNM) and limb-girdle muscular dystrophy type 2A (LGMD-2A), whereas fibs/PSWs were observed more often in desminopathy and facioscapulohumeral dystrophy (FSHD). Abnormal SA was commonly found in CNM (66.7%) and desminopathy (61.5%), occasionally in Duchenne (DMD) and Becker muscular dystrophy (BMD) (45.2% and 27.6%, respectively), but rarely in FSHD (14.9%) and LGMD-2A (12.0%). CONCLUSIONS: Abnormal SA probably occurs more frequently in disorders associated with structural changes in muscle fibers. Screening for SA may be a valuable tool for diagnosis of non-myotonic myopathies. Muscle Nerve 56: 427-432, 2017.

Diagnostic value of blink reflex in multisystem atrophy, progressive supranuclear palsy and Parkinson disease.

UNLABELLED: Abnormal blink reflex (BR) is a result of reticular brainstem pathways dysfunction and seems to be one of the features of brain degenerative disorders. The aim of the study was to estimate the diagnostic value of blink reflex in neurodegenerative diseases such as: multisystem atrophy (MSA), progressive supranuclear palsy (PSP) and Parkinson disease (PD). Material consisted of 99 patients with clinically probable MSA (51), PSP (28) and PD (20). MSA patients were divided into two subgroups, with dominant cerebellar (MSA-C) and parkinsonian signs (MSA-P). The mean age of patients was 64.9 years (47-79 years); males - 55.3%. Blink reflex was obtained in a typical way. RESULTS: The significant differences in mean values of blink reflex latencies between PD and other subgroups (MSA-P, MSA-C, PSP) were found, but all of them were in normal range. In individual patients with PD and PSP (50% and 18%, respectively) delayed R2 latencies were recorded. CONCLUSIONS: The most frequently abnormal blink reflexes, comparing the MSA, PSP and PD groups, were present in PD patients. We postulate that this may be explained by pathological influence of nigrostriatal pathway on the circuit linking the basal ganglia, cerebellum and brainstem.

Quality Control of Motor Unit Number Index (MUNIX) Measurements in 6 Muscles in a Single-Subject "Round-Robin" Setup.

BACKGROUND: Motor Unit Number Index (MUNIX) is a neurophysiological measure that provides an index of the number of lower motor neurons in a muscle. Its performance across centres in healthy subjects and patients with Amyotrophic Lateral Sclerosis (ALS) has been established, but inter-rater variability between multiple raters in one single subject has not been investigated. OBJECTIVE: To assess reliability in a set of 6 muscles in a single subject among 12 examiners (6 experienced with MUNIX, 6 less experienced) and to determine variables associated with variability of measurements. METHODS: Twelve raters applied MUNIX in six different muscles (abductor pollicis brevis (APB), abductor digiti minimi (ADM), biceps brachii (BB), tibialis anterior (TA), extensor dig. brevis (EDB), abductor hallucis (AH)) twice in one single volunteer on consecutive days. All raters visited at least one training course prior to measurements. Intra- and inter-rater variability as determined by the coefficient of variation (COV) between different raters and their levels of experience with MUNIX were compared. RESULTS: Mean intra-rater COV of MUNIX was 14.0% (±6.4) ranging from 5.8 (APB) to 30.3% (EDB). Mean inter-rater COV was 18.1 (±5.4) ranging from 8.0 (BB) to 31.7 (AH). No significant differences of variability between experienced and less experienced raters were detected. CONCLUSION: We provide evidence that quality control for neurophysiological methods can be performed with similar standards as in laboratory medicine. Intra- and inter-rater variability of MUNIX is muscle-dependent and mainly below 20%. Experienced neurophysiologists can easily adopt MUNIX and adequate teaching ensures reliable utilization of this method.

Does the MUNIX Method Reflect Clinical Dysfunction in Amyotrophic Lateral Sclerosis: A Practical Experience.

The aim of our study was to assess the usefulness of the MUNIX method in reflecting the clinical dysfunction in patients with amyotrophic lateral sclerosis (ALS), as well as to assess an intra-rater reproducibility of MUNIX. The study group consisted of a total of 15 ALS patients. The mean age of symptoms onset was 55 years, and the mean disease duration was 10 months. The muscle strength and patients' functional status were assessed according to the Medical Research Council (MRC) and by ALS functional rating scale revised (ALSFRS-R), respectively. The MUNIX was performed in 6 muscles: abductor pollicis brevis (APB), abductor digiti minimi (ADM), biceps brachii (BB), tibial anterior (TA), extensor digitorum brevis (EDB), and abductor hallucis (AH), unilaterally, at a less affected side. Both muscle-specific and global MRC and MUNIX scores were calculated. In 11 patients, the study protocol was repeated at least twice every 3 months. An additional testing of the intra-rater reliability was performed at the first visit.There were no significant differences between MUNIX test and re-test values in the APB, ADM, BB, TA, EDB, and AH muscles (P >0.05). The highest variability of the test-retest values was found in the BB muscle (7.53%). Although there was a significant test-retest difference in the global MUNIX score (P = 0.02), the variability of the results was as low as 1.26%. The MUNIX value correlated with the muscle-specific MRC score in ABP, ADM, TA, EDB and AH (P <0.05), and the global MUNIX values correlated with global MRC scores (P <0.05). There was also a significant correlation between the global MUNIX score and the clinical dysfunction measured by the ALSFRS-R scale (P <0.05). The global MUNIX showed a higher monthly decline (4.3%) as compared with ALFRS-R (0.7%) and the MRC global score (0.5%).This study confirms that the MUNIX method is a sensitive, reliable, and accurate tool reflecting both motor dysfunction and disease progression in ALS. We have found this approach to be more reliable and technically easier in distal muscles with less atrophy and a better strength.

Awaji criteria improves the diagnostic sensitivity in amyotrophic lateral sclerosis: A systematic review using individual patient data.

OBJECTIVE: To determine the utility of the Awaji criteria in diagnosing amyotrophic lateral sclerosis (ALS) and to propose a novel modification so as to enhance sensitivity based on results of individual patient data (IPD). METHODS: Individual patient data were available from 8 studies comparing the diagnostic accuracy of Awaji and revised El Escorial (rEEC) criteria. The sensitivity of a novel updated Awaji criteria, incorporating a "probable-laboratory supported" category, was also tested. RESULTS: Individual patient data were available from 1086 patients, consisting of 881 ALS and 205 patients with disorders mimicking ALS. Summary sensitivities based on random effects logistic regression modelling disclosed a higher sensitivity of the Awaji criteria (0.70, 95% confidence interval [CI] 0.51-0.83) and updated Awaji criteria (0.73, 95% CI 0.56-0.85) when compared to rEEC (0.58, 95% CI 0.48-0.68). Paired analysis revealed higher sensitivities of Awaji criteria in 4 studies, and of updated Awaji criteria in 7 studies, when compared to rEEC. CONCLUSION: Individual patient data analysis established a higher sensitivity of Awaji criteria when compared to rEEC. The updated Awaji criteria enhanced the diagnostic sensitivity in limb-onset ALS. SIGNIFICANCE: The updated Awaji criteria should be considered in clinical practice and future therapeutic trials.