Enhanced levels of fractalkine and HSP60 in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis patients.

Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disorder with a significant contribution of non-cell autonomous mechanisms to motor neuronal degeneration. Amongst a plethora of molecules, fractalkine (C-X3-C motif chemokine ligand 1), and Heat Shock Protein 60 (HSP60), are key modulators of microglial activation. The contribution of these molecules in Sporadic ALS (SALS) remains unexplored. To investigate this, fractalkine levels were estimated in Cerebrospinal fluid (CSF) of SALS patients (ALS-CSF; n = 44) by Enzyme-linked Immunosorbent Assay (ELISA) and correlated with clinical parameters including disease severity and duration. CSF HSP60 levels were estimated by Western blotting (ALS-CSF; n = 19). Also, CSF levels of Chitotriosidase-1 (CHIT-1), a microglia-specific neuroinflammatory molecule, were measured and its association, if any, with fractalkine and HSP60 was investigated. Both fractalkine and HSP60 levels were significantly elevated in ALS-CSF. Similar to our earlier observation, CHIT-1 levels were also upregulated. Fractalkine showed a moderate negative correlation with the ALS-Functional Rating Scale (ALSFRS) score indicating its significant rise in mild cases which plateaued in cases with high disease severity. However, no obvious correlation was found between fractalkine, HSP60, and CHIT-1. Our study hints that high fractalkine levels in mild cases might be conferring neuroprotection by combating microglial activation and highlights its importance as a novel therapeutic target for SALS. On the other hand, significantly enhanced levels of HSP60, a pro-inflammatory molecule, hint towards its role in accentuating microgliosis, although, it doesn't act synergistically with CHIT-1. Our study suggests that fractalkine and HSP60 act independently of CHIT-1 to suppress and accentuate neuroinflammation, respectively.

Apoptotic Factors and Mitochondrial Complexes Assist Determination of Strain-Specific Susceptibility of Mice to Parkinsonian Neurotoxin MPTP.

Identification of genetic mutations in Parkinson's disease (PD) promulgates the genetic nature of disease susceptibility. Resilience-associated genes being unknown till date, the normal genetic makeup of an individual may be determinative too. Our earlier studies comparing the substantia nigra (SN) and striatum of C57BL/6J, CD-1 mice, and their F1-crossbreds demonstrated the neuroprotective role of admixing against the neurotoxin MPTP. Furthermore, the differences in levels of mitochondrial fission/fusion proteins in the SN of parent strains imply effects on mitochondrial biogenesis. Our present investigations suggest that the baseline levels of apoptotic factors Bcl-2, Bax, and AIF differ across the three strains and are differentially altered in SN following MPTP administration. The reduction in complex-I levels exclusively in MPTP-injected C57BL/6J reiterates mitochondrial involvement in PD pathogenesis. The MPTP-induced increase in complex-IV, in the nigra of both parent strains, may be compensatory in nature. The ultrastructural evaluation showed fairly preserved mitochondria in the dopaminergic neurons of CD-1 and F1-crossbreds. However, in CD-1, the endoplasmic reticulum demonstrated distinct luminal enlargement, bordering onto ballooning, suggesting proteinopathy as a possible initial trigger.The increase in α-synuclein in the pars reticulata of crossbreds suggests a supportive role for this output nucleus in compensating for the lost function of pars compacta. Alternatively, since α-synuclein over-expression occurs in different brain regions in PD, the α-synuclein increase here may suggest a similar pathogenic outcome. Further understanding is required to resolve this biological contraption. Nevertheless, admixing reduces the risk to MPTP by favoring anti-apoptotic consequences. Similar neuroprotection may be envisaged in the admixed populace of Anglo-Indians.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

Neuro-Cardio-Autonomic Modulations in Children with Duchenne Muscular Dystrophy.

BACKGROUND AND OBJECTIVE: Duchenne muscular dystrophy (DMD) is a degenerative X-linked muscle disease. Death frequently results from complications in cardiopulmonary systems. Preclinical/early diagnosis of cardiac autonomic abnormalities may aid initiate cardioprotective therapy and enhance prognosis. METHODS: A cross sectional, prospective study of 38 DMD boys compared with 37 age-matched healthy controls was conducted. Lead II electrocardiography and beat-to-beat blood pressure were recorded to assess heart rate variability (HRV), blood pressure variability (BPV), and baroreceptor sensitivity (BRS) in a standardized environment. Data were analysed and correlated with disease severity and genotype. RESULTS: In the DMD group, the median age at assessment was 8 years [IQR 7-9 years], the median age at disease onset was 3 years [IQR, 2-6 years], and the mean duration of illness was 4 years [IQR, 2.5-5]. DNA sequencing showed deletions in 34/38 (89.5 %) and duplications in 4/38 (10.5%) patients. The median heart rate in DMD children was significantly higher [101.19 (Range, 94.71-108.49)] /min compared to controls [81 (Range, 76.2-92.76)] /min (p < 0.05). All the assessed HRV and BPV parameters were significantly impaired in DMD cases except for the coefficient of variance of systolic blood pressure. Further, BRS parameters were also significantly reduced in DMD, excluding alpha-LF. A positive correlation was found between alpha HF with age at onset and duration of illness. CONCLUSION: This study demonstrates a distinct early impairment of neuro-cardio-autonomic regulation in DMD. Simple yet effective non-invasive techniques such as HRV, BPV, and BRS may help identify cardiac dysfunction in a pre-clinical state, paving the way for early cardio-protective therapies and limiting disease progression in DMD patients.

Differences in Neuronal Numbers, Morphology, and Developmental Apoptosis in Mice Nigra Provide Experimental Evidence of Ontogenic Origin of Vulnerability to Parkinson's Disease.

Parkinson disease (PD) prevalence varies by ethnicity. In an earlier study, we replicated the reduced vulnerability to PD in an admixed population, using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-susceptible C57BL/6 J, MPTP-resistant CD-1 and their F1 crossbreds. In the present study, we investigated if the differences have a developmental origin. Substantia nigra was evaluated at postnatal days 2 (P2), P6, P10, P14, P18, and P22. C57BL/6 J mice had smaller nigra and fewer dopaminergic neurons than the CD-1 and crossbreds at P2, which persisted through development. A significant increase in numbers and nigral volume was observed across strains until P14. A drastic decline thereafter was specific to C57BL/6 J. CD-1 and crossbreds retained their numbers from P14 to stabilize with supernumerary neurons at adulthood. The neuronal size increased gradually to attain adult morphology at P10 in the resistant strains, vis-à-vis at P22 in C57BL/6 J. Accordingly, in comparison to C57BL/6 J, the nigra of CD-1 and reciprocal crossbreds possessed cytomorphological features of resilience, since birth. The considerably lesser dopaminergic neuronal loss in the CD-1 and crossbreds was seen at P2 and P14 and thereafter was complemented by attenuated developmental cell death. The differences in programmed cell death were confirmed by reduced TUNEL labelling, AIF, and caspase-3 expression. GDNF expression aligned with the cell death pattern at P2 and P14 in both nigra and striatum. Earlier maturity of nigra and its neurons appears to be better features that reflect as MPTP resistance at adulthood. Thus, variable MPTP vulnerability in mice and also differential susceptibility to PD in humans may arise early during nigral development.

Effect of Yoga and Physiotherapy on Pulmonary Functions in Children with Duchenne Muscular Dystrophy - A Comparative Study.

CONTEXT: Abnormal respiratory function is known to be detectable almost as soon as it can be measured reliably. Studies have identified the effect of respiratory muscle training as well as breathing exercises in improving pulmonary functions in children with Duchenne muscular dystrophy (DMD). AIMS: This study aims to identify the add-on effect of yoga over physiotherapy on pulmonary functions in children with DMD. SETTINGS AND DESIGN: One hundred and twenty-four patients with DMD were randomized to two groups. Group I received home-based physiotherapy and Group II received physiotherapy along with yoga intervention. MATERIALS AND METHODS: Pulmonary function test (PFT) was assessed before the intervention (baseline data) and at regular intervals of 3 months for a period of 1 year. STATISTICAL ANALYSIS USED: Normality was assessed using Shapiro-Wilk normality test. The baseline data were analyzed using Mann-Whitney U-test to identify the homogeneity. Repeated measures analysis of variance was used to assess significant changes in study parameters during the assessment of every 3 months, both within and between the two groups of patients. RESULTS: A total of 88 participants completed all the 5 assessments, with a mean age of 7.9 ± 1.5 years. PFT parameters such as forced vital capacity (FVC), peak expiratory flow rate, maximum voluntary ventilation (MVV), and tidal volume during maximum voluntary ventilation (MVt) demonstrated significant improvements in Group I. In Group II, FVC and MVt significantly improved from baseline up to 1 year, whereas MVV improved from baseline up to 9 months. Tidal volume did not show any changes in both the groups. CONCLUSIONS: The findings suggest that introduction of yoga with physiotherapy intervention at an early age can be considered as one of the therapeutic strategies in improving pulmonary functions in patients with DMD.

Chitotriosidase, a biomarker of amyotrophic lateral sclerosis, accentuates neurodegeneration in spinal motor neurons through neuroinflammation.

BACKGROUND: Cerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model. METHODS: CHIT-1 level in CSF samples from ALS (n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed (ALS, n = 56; non-ALS, n = 10 and normal-CSF, n = 45). Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA. FINDINGS: CHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 µmol/µl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons. CONCLUSIONS: CHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death.

A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord.

Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro techniques exceedingly relevant to study glial biology. However, obtaining optimal yield along with high purity has been challenging for microglial cultures. Here we present a simple protocol to establish enriched astroglial as well as microglial cultures from the neonatal rat spinal cord. This method results in highly enriched astroglial and microglial cultures with maximal yield.

Human placental extract ameliorates cytokine and cytokine receptor signaling in the rat hippocampus upon Benzo[a]Pyrene exposure.

Benzo[alpha]Pyrene (B[a]P) causes toxicity via Cytochrome P450 1A1 (CYP1A1) metabolic activity in the brain. Studies have shown that neuronal IL-2 and TNF-α are associated with the hippocampus development and regulation, but their association with the CYP1A1 activity remains unidentified. Limited action of human placental extract (HPE) in the activation of tissue repair and wound healing is known, but their role in B[a]P clearance in the hippocampus is not known so far. Our study has focused on two novel concepts: (1) association of CYP1A1 activity with the inflammatory response in the brain hippocampus and (2) role of HPE in the immunomodulatory mechanisms in the hippocampus upon B[a]P exposure at cytokine receptor and nuclear level. Intrathecal administration of different concentrations of B[a]P and HPE into male wistar rat pups has been conducted. An increased CYP1A1 activity was observed in the presence of 0.25 µM B[a]P alone but in case of HPE followed by 0.25 µM B[a]P, it was equal to control. Herein we report that 5 µl of 0.1 gm HPE followed by 0.25 µM B[a]P administration enabled down-regulation of IL-2 and TNF-α levels in the hippocampus thereby modulating TNFR2 and IL2Rγc signals via NF-κB activation. Besides, localization of IL-2, TNF-α, IL2Rγc, TNFR1 and TNFR2 in the CA1, CA3 and DG regions of the hippocampus are also depicted. Altogether, these findings will project the clinical importance of HPE in the neuroinflammation suppression in the hippocampus developed due to B[a]P toxicity.

Effect of Yoga as an Add-on Therapy in the Modulation of Heart Rate Variability in Children with Duchenne Muscular Dystrophy.

BACKGROUND: Duchene muscular dystrophy (DMD) is a progressive muscular disorder. Cardiac disorder is the second-most common cause of death in children with DMD, with 10%-20% of them dying of cardiac failure. Heart rate variability (HRV) is shown to be a predictor of cardio-autonomic function. Physiotherapy (PT) is advised for these children as a regular treatment for maintaining their functional status. The effect of yogic practices on the cardio-autonomic functions has been demonstrated in various neurological conditions and may prove beneficial in DMD. MATERIALS AND METHODS: In this study, 124 patients with DMD were randomized to PT alone or PT with yoga intervention. Home-based PT and yoga were advised. Adherence was serially assessed at a follow-up interval of 3 months. Error-free, electrocardiogram was recorded in all patients at rest in the supine position. HRV parameters were computed in time and frequency domains. HRV was recorded at baseline and at an interval of 3 months up to 1 year. Repeated-measures ANOVA was used to analyze longitudinal follow-up and least significant difference for post hoc analysis and P < 0.05 was considered statistically significant. RESULTS: In our study, with PT protocol, standard deviation of NN, root of square mean of successive NN, total power, low frequency, high-frequency normalized units (HFnu), and sympathovagal balance improved at varying time points and the improvement lasted up for 6-9 months, whereas PT and yoga protocol showed an improvement in HFnu during the last 3 months of the study period and all the other parameters were stable up to 1 year. Thus, it is evident that both the groups improved cardiac functions in DMD. However, no significant difference was noted in the changes observed between the groups. CONCLUSION: The intense PT and PT with yoga, particularly home-based program, is indeed beneficial as a therapeutic strategy in DMD children to maintain and/or to sustain HRV in DMD.

Cerebrospinal Fluid from Patients with Sporadic Amyotrophic Lateral Sclerosis Induces Degeneration of Motor Neurons Derived from Human Embryonic Stem Cells.

Disease modeling has become challenging in the context of amyotrophic lateral sclerosis (ALS), as obtaining viable spinal motor neurons from postmortem patient tissue is an unlikely possibility. Limitations in the animal models due to their phylogenetic distance from human species hamper the success of translating possible findings into therapeutic options. Accordingly, there is a need for developing humanized models as a lead towards identifying successful therapeutic possibilities. In this study, human embryonic stem cells-BJNHem20-were differentiated into motor neurons expressing HB9, Islet1, and choline acetyl transferase using retinoic acid and purmorphamine. These motor neurons discharged spontaneous action potentials with two different frequencies (< 5 and > 5 Hz), and majority of them were principal neurons firing with < 5 Hz. Exposure to cerebrospinal fluid from ALS patients for 48 h induced several degenerative changes in the motor neurons as follows: cytoplasmic changes such as beading of neurites and vacuolation; morphological alterations, viz., dilation and vacuolation of mitochondria, curled and closed Golgi architecture, dilated endoplasmic reticulum, and chromatin condensation in the nucleus; lowered activity of different mitochondrial complex enzymes; reduced expression of brain-derived neurotrophic factor; up-regulated neurofilament phosphorylation and hyperexcitability represented by increased number of spikes. All these changes along with the enhanced expression of pro-apoptotic proteins-Bax and caspase 9-culminated in the death of motor neurons.

Effects of craniopharyngioma cyst fluid on neurons and glial cells cultured from rat brain hypothalamus.

Craniopharyngiomas (CPs) are rare, epithelial tumors of the central nervous system (CNS) that could lead to manifestation of multiple post-operative symptoms, ranging from hormonal imbalance to obesity, diabetes, visual, neurological and neurocognitive impairments. CP is more frequent in children, and has been reported in middle aged adults as well. In fact, arterial laceration and/or brain stroke which may occur following the removal of some CPs is mainly due to calcification of that CPs along with strong attachments to the blood vessels. The dense oily fluid content of CPs is reported to cause brain tissue damage, demyelination and axonal loss in the hypothalamus; however, its exact effect on different cell types of CNS is still unexplored. In this study, we have collected CP cyst fluid (CCF) from mostly young patients during surgical removal and exposed it 9-10 days in vitro to the primary cultures derived from rat brain hypothalamus for 48 h. A gradual decline in cell viability was noted with increasing concentration of CCF. Moreover, a distinct degenerative morphological transformation was observed in neurons and glial cells, including appearance of blebbing and overall reduction of the cell volume. Further, enhanced expression of Caspase-3 in neurons and glial cells exposed to CCF by immunofluorescence imaging, supported by Western blot experiment suggest CCF induced apoptosis of hypothalamic cells in culture. In this study, we have demonstrated the deleterious effects of the cyst fluid on various cell types within the tumors originating region of the brain and its surroundings for the first time. Taken together, this finding could be beneficial towards identifying the region specific toxic effects of the cyst fluid and its underlying mechanism.

Effects of Chronic Carbamazepine Treatment on the ECG in Patients with Focal Seizures.

BACKGROUND AND OBJECTIVES: Several mechanisms have been proposed for the sudden unexpected death in epilepsy patients, such as cardiac arrhythmias, a decrease in heart rate variability and the use of anti-epileptic drugs (AEDs). Although carbamazepine is commonly used as an AED, the exact working mechanism of this drug as well as its effect on the heart are not completely understood. The aim of this study was to determine whether chronic carbamazepine therapy in patients with focal seizures and impaired awareness has an effect on the electrocardiogram (ECG). SUBJECTS AND METHODS: This cross-sectional study included 36 patients with focal seizures and impaired awareness treated for 12-32 months with carbamazepine monotherapy and 38 healthy volunteers. A 5-min modified three-electrode chest lead ECG with lead II configuration was recorded using LabChart 7 ECG software module at 1000-Hz sampling frequency. All data analysis was performed using custom-made Matlab 2015b scripts. ECGs of patients and controls were compared with respect to heart rate, time intervals and measures of short- and long-term variation. RESULTS: There were no significant differences in heart rate and ECG time intervals between the patient and control groups. Measures on short- and long-term variability also did not show any significant group differences. CONCLUSION: Our study shows that chronic use of carbamazepine as monotherapy does not have any significant effects on ECG time intervals or measures of short- and long-term variability.

Sporadic amyotrophic lateral sclerosis (SALS) - skeletal muscle response to cerebrospinal fluid from SALS patients in a rat model.

Skeletal muscle atrophy is the most prominent feature of amyotrophic lateral sclerosis (ALS), an adult-onset neurodegenerative disease of motor neurons. However, the contribution of skeletal muscle to disease progression remains elusive. Our previous studies have shown that intrathecal injection of cerebrospinal fluid from sporadic ALS patients (ALS-CSF) induces several degenerative changes in motor neurons and glia of neonatal rats. Here, we describe various pathologic events in the rat extensor digitorum longus muscle following intrathecal injection of ALS-CSF. Adenosine triphosphatase staining and electron microscopic (EM) analysis revealed significant atrophy and grouping of type 2 fibres in ALS-CSF-injected rats. Profound neuromuscular junction (NMJ) damage, such as fragmentation accompanied by denervation, were revealed by α-bungarotoxin immunostaining. Altered expression of key NMJ proteins, rapsyn and calpain, was also observed by immunoblotting. In addition, EM analysis showed sarcolemmal folding, Z-line streaming, structural alterations of mitochondria and dilated sarcoplasmic reticulum. The expression of trophic factors was affected, with significant downregulation of vascular endothelial growth factor (VEGF), marginal reduction in insulin-like growth factor-1 (IGF-1), and upregulation of brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF). However, motor neurons might be unable to harness the enhanced levels of BDNF and GDNF, owing to impaired NMJs. We propose that ALS-CSF triggers motor neuronal degeneration, resulting in pathological changes in the skeletal muscle. Muscle damage further aggravates the motor neuronal pathology, because of the interdependency between them. This sets in a vicious cycle, leading to rapid and progressive loss of motor neurons, which could explain the relentless course of ALS.This article has an associated First Person interview with the first author of the paper.

Influence of age and gender on the function of postganglionic sympathetic sudomotor axons.

BACKGROUND: The quantitative sudomotor axon reflex test (QSART) is an autonomic function test to evaluate the function of postganglionic sympathetic sudomotor axons. The QSART is used for research and in clinical assessment of various neurological diseases, but few studies have assessed the influence of age, gender and reported a normative range. We assessed the influence of age and gender on QSART parameters. METHODS: We recruited 61 healthy volunteers (41 men and 20 women) after obtaining written informed consent. The QSART was recorded as per standard protocol after iontophoretic stimulation (using acetylcholine) for 5 minutes. We analysed the sweat response to obtain total sweat production, rate of sweat production and latency time from the start of stimulation to sweat response. We assessed these parameters in the right medial forearm, right proximal leg, right distal leg and right proximal foot. RESULTS: Men had significantly higher evoked sweat volume and sweat latency period compared to women. A positive correlation was observed between age and evoked total sweat volume. An inverse correlation was noted between age and evoked sweat latency period. CONCLUSION: Postganglionic sudomotor function increased significantly with age. Men had significantly higher sweat volume suggesting sympathetic predominance. These results might help establish normative data for the Indian population.

VEGF alleviates ALS-CSF induced cytoplasmic accumulations of TDP-43 and FUS/TLS in NSC-34 cells.

Cytoplasmic mislocalisation and aggregation of TDP-43 and FUS/TLS proteins in spinal motor neurons contribute to the pathogenesis of the highly fatal disorder amyotrophic lateral sclerosis (ALS). We investigated the neuroprotective effect of VEGF on expression of these proteins in the motor neuronal cell line NSC-34 modelled to reminisce sporadic form of ALS. We studied the expression of TDP-43 and FUS/TLS proteins after exposure to ALS-CSF and following VEGF supplementation by quantitative confocal microscopy and electron microscopy. ALS-CSF caused cytoplasmic overexpression of both the proteins and stress-granule formation in the cells. These alterations were alleviated by VEGF supplementation. The related ultrastructural changes like nuclear membrane dysmorphism and p-bodies associated changes were also reversed. However the protein expression did not completely translocate to the nucleus, as some cells continued to show to cytoplasmic mislocalisation. Thus, the present findings indicate that VEGF alleviates TDP43 and FUS pathology by complimenting its role in controlling apoptosis and reversing choline acetyl transferase expression. Hence, VEGF appears to target multiple pathogenic processes in the neurodegenerative cascade of ALS.

Brain-Derived Neurotrophic Factor Facilitates Functional Recovery from ALS-Cerebral Spinal Fluid-Induced Neurodegenerative Changes in the NSC-34 Motor Neuron Cell Line.

BACKGROUND: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. OBJECTIVE: To evaluate the protective role of BDNF in a model of sporadic ALS patients. METHODS: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. RESULTS: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. CONCLUSION: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.

Astroglia acquires a toxic neuroinflammatory role in response to the cerebrospinal fluid from amyotrophic lateral sclerosis patients.

BACKGROUND: Non-cell autonomous toxicity is one of the potential mechanisms implicated in the etiopathogenesis of amyotrophic lateral sclerosis (ALS). However, the exact role of glial cells in ALS pathology is yet to be fully understood. In a cellular model recapitulating the pathology of sporadic ALS, we have studied the inflammatory response of astroglia following exposure to the cerebrospinal fluid from ALS patients (ALS-CSF). METHODS: Various inflammatory markers including pro-inflammatory and anti-inflammatory cytokines, COX-2, PGE-2, trophic factors, glutamate, nitric oxide (NO), and reactive oxygen species (ROS) were analyzed in the rat astroglial cultures exposed to ALS-CSF and compared with the disease control or normal controls. We used immunofluorescence, ELISA, and immunoblotting techniques to investigate the protein expression and real-time PCR to study the messenger RNA (mRNA) expression. Glutamate, NO, and ROS were estimated using appropriate biochemical assays. Further, the effect of conditioned medium from the astroglial cultures exposed to ALS-CSF on NSC-34 motor neuronal cell line was detected using the MTT assay. Statistical analysis was carried out using one-way ANOVA followed by Tukey's post hoc test, or Student's t test, as applicable. RESULTS: Here, we report that the ALS-CSF enhanced the production and release of inflammatory cytokines IL-6 and TNF-α, as well as COX-2 and PGE-2. Concomitantly, anti-inflammatory cytokine IL-10 and the beneficial trophic factors, namely VEGF and GDNF, were down-regulated. We also found impaired regulation of glutamate, NO, and ROS in the astroglial cultures treated with ALS-CSF. The conditioned medium from the ALS-CSF exposed astroglial cultures induced degeneration in NSC-34 cells. CONCLUSIONS: Our study demonstrates that the astroglial cells contribute to the neuroinflammation-mediated neurodegeneration in the in vitro model of sporadic ALS.

Heart Rate Variability in Children with Attention-Deficit/Hyperactivity Disorder: A Pilot Study.

BACKGROUND: Attention deficit/hyperactivity disorder (ADHD) is a common childhood neuropsychiatric disorder. Autonomic nervous system plays a vital role in attention, self-regulation, emotional stability and social affiliation, which are affected in ADHD. The prefrontal cortex, which is vital for attention, motor control, emotional regulation and higher order autonomic control, is hypofunctional in ADHD. In addition, catecholamine dysregulation is there. PURPOSE: We hypothesized that there is autonomic dysfunction: reduction in overall heart rate variability (HRV) and sympathovagal imbalance in children with ADHD. METHODS: Study criteria were drug-naïve ADHD children who were 7-12 years of age of either gender who fulfilled DSM-IV criteria for ADHD and did not have any associated comorbid psychiatric/neurological/medical disorders. Two hundred and seventy ADHD children were screened out of which only 12 were found eligible and 10 participated. Sample size was 20 (cases = 10, age- and gender-matched healthy controls = 10). Short-term HRV of both time and frequency domains were assessed by recording lead II electrocardiogram after using Tell-Show-Do, a behavior shaping technique. Comparison between groups was done using Mann-Whitney and Wilcoxon test. Demographic variables like age, height, weight and body mass index were similar between groups. RESULTS: Among time domain parameters, SD of all NN intervals, square root of the mean of the sum of squares of differences between adjacent NN intervals and percentage of count of number of pairs of adjacent NN intervals differing by more than 50 ms were reduced in ADHD group with p < 0.05. Among frequency domain parameters, total power was reduced in ADHD group with p < 0.05, high frequency power (HF) was reduced in ADHD group with p < 0.01 and low frequency power to HF ratio was higher in ADHD group with p < 0.01. CONCLUSION: There is autonomic dysfunction in children with ADHD - reduction in overall HRV with sympathovagal imbalance with sympathetic dominance.

Influence of hydrotherapy on clinical and cardiac autonomic function in migraine patients.

BACKGROUND: Migraine is associated with autonomic symptoms. The growing body of literature suggests that the dysfunctional autonomic nervous system might play a pivotal role in the pathogenesis of migraine. Thermal therapies have been hypothesized to modulate these changes and alleviate pain. However, data regarding the efficacy of hydrotherapy in migraine remain scant. We evaluated the effect of add on hydrotherapy procedure (a hot arm and foot bath with ice massage to head) in migraine patients. METHODS: Forty chronic migraine patients fulfilling the International Classification of Headache Disorders II criteria were recruited from the neurology outpatient clinic. Patients were randomized to receive either hydrotherapy plus conventional pharmacological care (n = 20) or conventional medication only (n = 20). Hydrotherapy group received treatment with hot arm and foot bath (103°F to 110°F) and ice massage to head daily for 20 min for 45 days. Patients were assessed using headache impact test (HIT), visual analog scale for pain and cardiac autonomic function by heart rate variability (HRV) before and after intervention period. RESULTS: There was a significant decrease in HIT score, frequency, and intensity of headaches following treatment in both the groups. However, it was more evident in add on hydrotherapy group compared to pharmacological treatment alone group. There was also significant improvement in the HRV parameters. In particular, there was a significant decrease in heart rate (P = 0.017), increase in high frequency (HF) (P = 0.014) and decrease in low frequency/HF ratio (P = 0.004) in add on hydrotherapy group. CONCLUSION: Our study shows that add on hydrotherapy enhanced the vagal tone in addition to reducing the frequency and intensity of headaches in migraine patients.