THEME 11 IMAGING, ELECTROPHYSIOLOGY AND MARKERS OF DISEASE PROGRESSION

P191 TRANSCRANIAL SONOGRAPHY OF THE SUBSTANTIA NIGRA IN ALS: PILOT DATA

FATHINIA P, KASSUBEK J, LUDOLPH AC

University Hospital Ulm, Ulm, Germany

E-mail address for correspondence: [email protected]

Keywords: transcranial sonography, substantia nigra, symptomatic therapy

Background: Transcranial sonography is a fast and fully non-invasive technique for the depiction of the basal ganglia, as already extensively assessed in Parkinson´s disease.

Objectives: The idea of this pilot study was to examine if there is a link between morphological alterations, ie hyperechogenicity of the substantia nigra and ALS as a correlate of associations between the clinical finding of coexistent rigidity in motor neuron disorders.

Methods: Thirty-three patients (aged 38 to 78, 17 m/16 f) with the diagnosis of possible, probable or definite ALS were examined with trancranial sonography. The hyperechogenic area of the mesencephal brainstem was measured planimetrically. Thus, hyperechogenicity of the substantia nigra was defined by an increased size of this region compared both with literature (1) values and compared with a healthy control group (18 patients, aged 27 to 75, 10 m/8 f). We defined hyperechogenicity as a planimetric measurement result of more than 0.20 cm², as also used in Parkinson´s disease.

Results: Twenty-three ALS patients showed a significant bilateral hyperechogenicity of the substantia nigra with planimetric measurements over 0.25 cm². Five patients showed no changes compared to controls and five other patients could not be assessed due to insufficient bone window. The controls had a median hyperechogenic area size of 0.1 cm².

Discussion: Out of 33 patients with the diagnosis of ALS examined via transcranial sonography, 23 patients showed a hyperechogenicity of the substantia nigra. These pilot results will await confirmation by examination in a larger patient collective.

Conclusions: By sonographic substantia nigra assessment, it might be possible to establish a correlation between morphological alterations of the substantia nigra and the symptomatic therapeutic value of L-Dopa in ALS.

Reference:

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P192 QUANTITATIVE SENSORY TESTING DEMONSTRATES C-FIBRE, A-δ FIBRE, AND A-β-FIBRE INVOLVEMENT IN ALS

PONFICK M, KLUG R, GDYNIA H-J, GASTL R, KASSUBEK J, SPERFELD A-D, LUDOLPH AC

University of Ulm, Department of Neurology, Ulm, Baden-Württemberg, Germany

E-mail address for correspondence: [email protected]

Keywords: quantitative sensory testing, small-fibre neuropathy, somatosensory nerve system

Background: Although the hallmark of ALS is a progressive degeneration of the central and peripheral motor system, variable deficits of the somatosensory nervous system have repeatedly been described.

Objective: The aim of this study was to examine the potential of quantitative sensory testing (QST) in order to further substantiate a sensory involvement in ALS.

Methods: Twenty-one patients with ALS (mean age 62.4±10.7 years, mean disease duration 41.9±34.6 months, mean ALSFRS-R 30.95±10.9) were tested with QST using a standardized and validated protocol.

Results: Clinically, only 9/21 (0.43, 95 CI: 0.23–0.66) patients showed sensory disturbances, whereas the QST revealed distinct abnormalities in 20/21 (0.95, 95 CI: 0.76–1.0) patients. Nineteen patients (0.90, 95 CI: 0.70–0.99; 2x 19/21) showed abnormalities in at least one parameter which are representing C-fibre and A-δ fibre function. Twelve patients (0.57, 95 CI: 0.34–0.78) presented at least one pathologic value in A-β-fibre parameter and fourteen (0.67, 95 CI: 0.43–0.85) showed central pain processing disorders.

Discussion: By using QST in ALS patients, we detected (sometimes subtle) somatosensory nervous system involvement in the majority of patients. Currently, further investigations concerning histological examinations of skin nerve biopsies, in order to analyse potential small-fibre involvement are ongoing.

Conclusions: The QST seems to be an appropriate method to detect sensory fibre involvement in ALS.

P193 SLOW SACCADES IN BULBAR-ONSET MOTOR NEURONE DISEASE

DONAGHY C¹, PINNOCK R¹, FORBES R¹, HARDIMAN O², PATTERSON V¹, MCGIVERN CR¹, GIBSON MJ¹

¹Royal Victoria Hospital, Belfast, United Kingdom, ²Beaumont Hospital, Dublin, Ireland

E-mail address for correspondence: [email protected]

Keywords: saccades, ocular fixation, PSP

Background: Current findings suggest that eye movement abnormalities in ALS relate to frontal lobe impairment. Numerous case reports, however, describe slow saccades and supranuclear gaze palsies in patients with MND often associated with bulbar-onset disease. We performed a study of saccades and ocular fixation in patients with MND to examine for any differences between bulbar-onset, spinal-onset ALS and controls. We then compared the results for bulbar-onset ALS with a group of patients with PSP.

Methods: Forty-four ALS patients, 45 controls and 7 PSP patients were included. Reflexive horizontal saccades (latency and speed) and ocular fixation (saccadic intrusion amplitude and frequency) were examined using infra-red oculography. A Saccadic Intrusion (SI) Index was developed by the authors representing saccadic intrusion frequency and amplitude.

Results: Saccades were found to be slower in bulbar-onset compared to spinal-onset patients and controls (P=0.03, P=0.05). PSP patients had significantly slower saccades compared to controls (P=0.006). SI frequency was greater in PSP and bulbar-onset ALS patients compared to controls (P=0.006, 0.015). Similarly, SI Index was greater in PSP and bulbar-onset ALS patients compared to controls (P=0.01, 0.002). Although not statistically significant, PSP patients had increased SI freq, SI amplitude, SI Index and slower saccades compared to bulbar-onset ALS patients.

Conclusions: This is the first study to highlight the presence of slow saccades in bulbar-onset MND. It is likely that a spectrum exists, ranging from normal saccade speed to the presence of a gaze palsy. This theory is consistent with the similarities found in the eye movement profile of PSP and bulbar-onset ALS patients. A longitudinal study would be of great interest to examine the potential of eye movements as a biomarker for ALS disease progression. In addition, SI Index appears to be a sensitive measure of ocular fixation abnormality that could be employed in further research.

P194 REDUCED MOTOR NETWORK CONNECTIVITY DURING REST IN ALS

JELSONE-SWAIN L¹, GRUIS K², HOVATTER R¹, SEIDLER R^4,5, FLING B⁵, WELSH R^1,3

¹Department of Radiology, ²Department of Neurology, ³Department of Psychiatry, ⁴Department of Psychology, ⁵Division of Kinesiolog; University of Michigan, Ann Arbor, MI, United States

E-mail address for correspondence: [email protected]

Keywords: resting state, fcMRI, motor cortex

Background: Neurodegenerative diseases do not manifest randomly, but affect disease-specific cortical neural networks. In ALS, observations of altered structure and function in the sensorimotor network have been reported, however the intrinsic global functioning of upper motor neurons during early disease stages is poorly understood. Furthermore, it is not known how functionally connected nodes within this network change with disease progression. Identifying these changes with resting-state functional connectivity magnetic resonance imaging (fcMRI) has important implications in understanding ALS pathophysiology and possible diagnostic implications.

Objectives: Using fcMRI during rest, our goal was to examine cortical coupling: 1) between primary motor cortices (M1) and between nodes of the entire sensorimotor network; 2) examine the sensorimotor network in high-functioning patients to delineate neural changes that may precede moderate-severe symptom presentation; and 3) identify longitudinal functional changes in these networks.

Methods: Twenty patients with ALS (11 males, < age>=59 years) within 24 months of diagnosis, and 20 age/sex-matched healthy volunteers (13 males, < age>=58 years) were scanned at the University of Michigan's MRI laboratory on a GE 3T magnet. Of these 20, nine longitudinal ALS participants were scanned again an average of nine months later. fcMRI image acquisition lasted six minutes during rest. Two network analyses were performed to directly examine: 1) interhemipsheric M1 coupling and 2) whole sensorimotor network connectivity. M1 coupling: Anatomical masks were created along the precentral gyrus in each hemisphere and then segmented into forty regions of interest (ROI), thus increasing sensitivity to discretely localized regions that may be affected by ALS. Sensorimotor connectivity: Single ROIs in the dorsal and ventral premotor cortex, supplementary motor area (SMA), pre-SMA, M1, and somatosensory cortex in each hemisphere were created. Correlation strength and number of significant connections between ROIs for each analyses were compared between groups and scanning sessions.

Results: The ALS group showed significantly less interhemispheric M1 connectivity than the healthy control group (P<0.01). A group x ROI interaction (P<0.00001) indicated that decreased connectivity was more pronounced in dorsal regions of M1. Within subjects analysis of the ALS group resulted in significantly reduced interhemispheric correlations at their subsequent scanning session (P<0.005). Examining the sensorimotor network, the number of statistically-significant connections was less in the ALS group, with 31 connections present in the healthy control group, 28 connections in the ALS group at time-point-one and 23 connections at time-point-two.

Discussion and conclusion: Results from the current study indicate that functional neural changes are occurring early, before moderate symptom presentation, and are rapidly progressing. Furthermore, the pattern of decreased network connectivity may be more specific to dorsal M1 locations, which correspond to limb and trunk regions of the body. Further study is needed to place these findings in the context of the pathophysiology of the disease.

P195 MOTOR NETWORK DEGENERATION IN ALS: A STRUCTURAL AND FUNCTIONAL CONNECTIVITY STUDY

VERSTRAETE E, VAN DEN HEUVEL MP, MANDL RC, VELDINK JH, VAN DEN BERG LH

UMC Utrecht, Utrecht, Netherlands

E-mail address for correspondence: [email protected]

Keywords: cortical thickness, diffusion tensor imaging, resting-state functional MRI

Background: Amyotrophic lateral sclerosis (ALS) is known to primarily affect motor neurons. The pattern of neurodegeneration and how it affects the central motor network as a whole is however, unknown.

Objectives: To integrate structural and functional imaging measures on the motor network in patients with ALS and healthy controls.

Methods: Twelve patients with ALS and twelve matched controls were studied. Cortical thickness measurements and diffusion tensor imaging (DTI) were carried out on crucial motor tracts, including the corticospinal tracts (CST) and the corpus callosum. Furthermore, these structural measures were combined with assessment of functional connectivity of the motor network based on resting state fMRI.

Results: Cortical thinning was observed in the primary motor areas in ALS patients compared to controls and was found to be associated with disease progression. Moreover, FA values were found significantly reduced in the corpus callosum and in the rostral part of the CST. Overall functional organisation of the motor network was not significantly altered, but the level of functional connectedness in patients with ALS was found to be associated with disease progression rate.

Discussion and conclusions: We demonstrate central motor network deterioration in ALS together with clinical implications of the functional connectedness of the motor network. These data corroborate the spread of disease along the functionally and structurally linked neural structures of the motor network.

P196 UPPER MOTOR NEURON ABNORMALITIES ON CONVENTIONAL BRAIN MRI IN ALS PATIENTS: CLINICAL FEATURES AND NATURAL HISTORY

MATTE G, PIORO E

Cleveland Clinic, Cleveland, OH, United States

E-mail address for correspondence: [email protected]

Keywords: neuroimaging, phenotype, disease progression

Background: Routine T2 and proton density (PD) sequences on conventional brain MRI (cMRI) occasionally reveal abnormalities in upper motor neuron (UMN) regions of ALS patients as corticospinal (CST) hyperintensity and primary motor cortex (PMC) hypointensity. The clinical, pathological, and prognostic significance of these changes is unknown, and distribution of CST hyperintensities is poorly defined.

Objective: Define the phenotype and prognosis associated with CST hyperintensity and/or PMC hypointensity on T2 and PD of cMRI sequences in ALS patients.

Methods: Clinical data were retrospectively reviewed from 112 patients diagnosed with ALS at the Cleveland Clinic who underwent brain MRI. Five millimeter-thick transverse brain images were obtained at 1.5T using fast spin-echo parameters: TR=3750 ms, TE=80, 20 ms.

Results: Forty six of 112 patients (41%) had PMC hypointensity and/or CST hyperintensity on a single cMRI scan performed 26 months (mean) after symptom onset. Patients with either of these changes were younger at symptom onset (53.1 vs. 58.4 years, P=0.02) and tended to be males (32/46 vs. 33/66, P=0.052). Patients with cMRI changes typically displayed predominant UMN phenotype (40/46 vs. 37/66, P=0.001), and rarely had an exclusively lower motor neuron phenotype (1/46 vs. 12/66, P=0.014). Patients with CST hyperintensity were younger at symptom onset than patients with isolated PMC hypointensity (50.9 vs. 60.0 years, P=0.03) or no MRI abnormalities (50.9 vs. 58.4 years, P=0.005). Patients with CST hyperintensity had a shorter survival than patients with PMC hypointensity (3.5 vs. 7.0 years, P=0.004). When present, CST hyperintensity occurred at the cerebral peduncle (CP) level in 35/35 patients and in a contiguous fashion if visible rostrally. Patients with both PMC hypointensity and CST hyperintensity were more likely to have hyperintensity extending to the immediate subcortical white matter than patients with only CST hyperintensity (10/17 vs. 3/18, P=0.015). However, these two groups were similar in clinical phenotype and prognosis.

Discussion: We demonstrate in a large cohort of ALS patients with long-term clinical follow-up that presence of these cMRI changes is associated with characteristic clinical features. CST hyperintensity with or without PMC hypointensity is associated with a more aggressive UMN phenotype, while PMC hypointensity alone identifies a disease course resembling that observed in the absence of cMRI changes. CST hyperintensity is most likely seen at the CP, possibly because of the compactness of fibers here, but its presence more rostrally may represent either retrograde UMN degeneration or a different pathogenic process.

Conclusions: ALS patients with CST hyperintensity and/or PMC hypointensity develop UMN-predominant disease at a younger age than patients without such cMRI changes. Whether this indicates differences in pathogenic mechanisms or UMN degeneration requires further study. Identifying ALS subgroups with distinct clinical and prognostic features using cMRI may be useful for patient stratification in ALS clinical trials.

P197 VOXEL-BASED MORPHOMETRY AND FUNCTIONAL MAGNETIC RESONANCE IMAGING STUDY IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS WITH BEHAVIORAL IMPAIRMENT

TROJSI F^1,2, ESPOSITO F^2,3, SAGNELLI A¹, PACCONE A², CORBO D², PICCIRILLO G¹, TEDESCHI G^1,2, MONSURRò MR¹

¹Department of Neurological Sciences, Second University of Naples, Naples, Campania, Italy, ²Neurological Institute for Diagnosis and Care “Hermitage Capodimonte”, Naples, Campania, Italy, ³Department of Neuroscience, University of Naples “Federico II”, Naples, Campania, Italy

E-mail address for correspondence: [email protected]

Keywords: resting-state functional magnetic resonance imaging (RS-fMRI), voxel-based morphometry, default-mode network (DMN)

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease, mainly characterized by the progressive degeneration of upper and lower motor neurons, but does not spare extra-motor areas, causing cognitive and behavioral syndromes.

To better investigate structural and functional abnormalities in ALS, we used an optimized version of voxel-based morphometry (VBM) analysis combined to functional resting-state fMRI data in a population of behaviour impaired patients.

Resting-state functional magnetic resonance imaging was performed at 3 Tesla on 20 ALS patients with frontal dysfunction and 20 age- and sex-matched healthy volunteers. Resting-state network maps, extracted with independent component analysis and group-level statistical analyses, were performed to detect disease and disease-by-age interaction effects. Using the high quality 3D-T1 whole-head scans available for all 40 subjects, we conducted an optimized voxel-based morphometric analysis, importing 3D-T1 scans to the SPM8 software package. Then, to allow voxel-by-voxel comparisons with network maps in the same anatomical space, the series of segmented grey matter (GM) images were transformed to the Talairach space.

The sensori-motor network showed significant disease effects, with signal suppression in patients in the primary and the supplementary motor cortices, principally in the left hemisphere. The same was visible frontally in the right fronto-parietal network, possibly reflecting the patients’ frontal dysfunction. The default-mode network showed no group differences, but a significant disease-by-age interaction in the posterior cingulate cortex, where signals positively correlated with age and disease duration and negatively correlated with the functional rating scale score in patients. Similar effects were detected within the right fronto-parietal network. Compared with controls, ALS patients had significant clusters of locally reduced GM density (P < 0.05) in the left premotor and right fronto-parietal cortex.

We observed no spots of reduced GM in ALS patients within the default-mode network, suggesting that possible compensation mechanisms linked to the observed disease-by-age interactions in the cognitive networks are active in regions with no ongoing structural degeneration.

Therefore, the disease-by-age interaction in the default-mode and right fronto-parietal networks unravels a possible mechanism of compensation between motor and extra-motor systems. Given the striking spatial contiguity of disease effects in default and sensorimotor networks connectivity and gray matter densities values, we can speculate that the suppression of the within-network RS-fMRI signals may be an earlier marker of the structural degeneration and not vice versa, concluding with the intriguing finding that the mechanism of functional networks’ rearrangements anticipates in ALS the atrophic degeneration process.

P198 WHITE MATTER TRACT DAMAGE AND COGNITIVE DYSFUNCTION IN ALS

PETTIT L, BASTIN M, ABRAHAMS S

University of Edinburgh, Edinburgh, Midlothian, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: imaging, cognitive, dysfunction

Background: Cognitive dysfunction in non-demented amyotrophic lateral sclerosis (ALS) has been consistently demonstrated. Tests of verbal fluency (VF) are the most commonly reported impairments and have been associated with reduced white matter volume in frontotemporal areas. Recent developments in magnetic resonance imaging (MRI) techniques allow the investigation of white matter structure and integrity.

Objectives: To investigate the extent and location of white matter damage in ALS patients and its relationship to cognitive dysfunction.

Method: Conventional and Diffusion Tensor (DT) magnetic resonance imaging data were acquired from 8 ALS patients and 8 healthy age-matched controls. A measure of mean diffusivity (MD) was extrapolated from the data and compared between groups using Tract Based Spatial Statistics (TBSS). Cognitive functioning in the ALS group was assessed by neuropsychological tests of verbal fluency, the Brixton spatial awareness test, and the graded naming test (GNT).

Results: Contrast analyses revealed significantly (P<0.05, corrected for multiple comparisons) higher MD in patients compared to controls in extensive prefrontal white matter regions, including regions through which run: the inferior longitudinal fasciculus (connecting the temporal and occipital lobes), the arcuate fasciculus (connecting temporoparietal and frontal areas), the uncinate fasciculus (connecting the limbic system to orbitofrontal cortex), and the cingulum. In addition, ALS patients had significantly higher MD in the internal and external capsules, as well as frontal association fibres running into superior frontal gyrus (BA 10) and dorsolateral prefrontal cortex (BA 9, 44, and 46). Results from the cognitive tasks indicated that the ALS group performed worse than controls in spoken verbal fluency (Z score = −1.8 compared to control mean). Four ALS patients had Z scores greater than −1.96 suggesting that half of the patients were significantly impaired on fluency. The ALS group performed comparably to control norms in the GNT and Brixton tests, however patients’ Brixton scores were variable with two patients in the abnormal/ poor range and three patients in the superior range.

Discussion: ALS patients exhibited damage to multiple white matter tracts, as indicated by high mean diffusivity. There was a preponderance of prefrontal involvement, with tracts leading to dorsolateral, superior/medial, and orbitofrontal regions showing poor integrity. Patients within this group were found to be impaired on verbal fluency, suggesting that white matter integrity may underlie this cognitive dysfunction. Impoverished white matter integrity may be a symptom of spreading neuronal pathology or Wallerian degeneration following neuronal loss and requires further investigation.

Conclusions: The current investigation provides strong evidence of damage to white matter tracts in ALS, and suggests that white matter integrity may underpin the pattern of cognitive impairment demonstrated in a proportion of patients.

P199 NEURORADIOLOGICAL CHARACTERISATION OF EXTRA MOTOR CORTEX PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS

BEDE P^1,2, BOKDE A^1,2, JORDAN N², ELAMIN M^1,2, FAGAN A³, HARDIMAN O^1,2

¹Trinity College, Dublin, Ireland, ²Beaumont Hospital, Dublin, Ireland, ³St James Hospital, Dublin, Ireland

E-mail address for correspondence: [email protected]

Keywords: MRI, cognition, frontotemporal dementia

Background: Amyotrophic Lateral Sclerosis is a complex multisystem disorder with non-motor manifestations: neuropsychological and behavioural deficits. ALS with Frontotemporal Dementia (ALS-FTD) is increasingly recognized as a distinct phenotype of Motor Neuron Disease with distinguishing clinical, neuroradiological and neuropsychological features. Behavioural and cognitive deficits in ALS may precede motor symptoms, are challenging to manage and have a significant impact on survival and the quality of life of carers and patients.

Aims: The aim of this study is to determine the extent of frontotemporal pathology in ALS patients with no overt cognitive impairment and to describe the Magnetic Resonance Imaging characteristics of the ALS-FTD complex.

Methods: Thirty age matched healthy controls and 30 ‘definite’ ALS patients according to the El Escorial criteria have been recruited. Participants are assessed by a comprehensive battery of neuropsychological tests and undergo 3 Tesla high resolution magnetic resonance neuroimaging. The neuropsychological battery includes the Boston naming test, Raven's standard progressive matrices, Logical Memory tests, the California verbal learning test, Verbal paired associates, the Rey Complex Figure test, the Brixton spatial anticipation test, Digit span, Verbal fluency tests, Category Fluency, the Wechsler test of adult reading, The colour word task –Stroop test, the Hospital Anxiety and Depression Scale and the Frontal Systems Behaviour Scale (FrSBe). The neuroimaging protocol includes: Voxel Based Morphometry (VBM) based on high resolution structural imaging data, Magnetic Resonance Spectroscopy (MRS), Diffusion Tensor Imaging (DTI) and Resting state Functional MRI (rfMRI). A routine Fluid-attenuated inversion recovery (FLAIR) sequence is used for the identification of underlying cerebrovascular disease to exclude patients from the research study.

Results: Five ALS patients fulfilled the Neary Criteria for Frontotemporal dementia. Fifteen ALS patients scored within normal range in all of the neuropsychological domains when compared to a pool of age matched healthy controls, and were categorized as ALS patients with no cognitive deficits. Ten ALS patients demonstrated executive dysfunction on neuropsychological testing. Voxel based morphometry (VBM) analysis demonstrated considerable differences in grey matter volumes between ALS-FTD and ALS patients with no cognitive deficits. Comparative statistical maps highlighted significant differences (P<0.05) in the left anterior temporal lobe, ventral medial frontal lobe and parahippocampal regions. ALS patients with no cognitive deficits showed grey matter volume changes in the hippocampus, left dorsolateral prefrontal cortex, cerebellum, posterior cingulate and in the in right superior temporal gyrus when compared to healthy controls.

Conclusions: The results underscore the heterogeneity of extra motor involvement within the ALS spectrum. The study demonstrates the unique radiological attributes of the ASL-FTD complex. However, ALS patients with no cognitive or behavioural deficits on formal neuropsychological testing also show signs of extensive extra motor cortex atrophy on a group level.

P200 CORPUS CALLOSUM INVOLVEMENT IS A CORE FEATURE OF ALS, AND MAY REFLECT EARLY INTER-HEMISPHERIC SPREAD OF PATHOLOGY

FILIPPINI N, DOUAUD G, MACKAY C, KNIGHT S, TALBOT K, TURNER M

Oxford University, Oxford, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: corpus callosum, diffusion tensor imaging, biomarker

Background: Focus of onset and spread of pathology throughout upper (UMN) and lower motor neuron (LMN) compartments in ALS are poorly understood. Diffusion tensor imaging (DTI) is an established and sensitive MRI application for the non-invasive detection of white matter (WM) pathology, but biomarkers to improve diagnosis and therapeutic monitoring in ALS must accurately reflect the inherent clinical and prognostic heterogeneity.

Objectives: We sought to establish a core signature of cerebral WM change in heterogeneous cases of ALS with variable UMN involvement clinically, using unbiased whole-brain DTI, that might also inform onset and spread of pathology.

Methods: High-field 3T DTI was applied (using whole-brain Tract-based Spatial Statistics, TBSS) to a group of 24 unselected heterogeneous ALS patients, in comparison with very closely age and gender-matched healthy controls. Patients were scored for clinical UMN involvement and disability (using the revised ALS Functional Rating Scale, ALSFRS-R). Voxel-based morphometry of T1 images was also undertaken to explore any associated grey matter (GM) atrophy.

Results: A consistent, highly significant reduction in fractional anisotropy (FA) was demonstrated in the corpus callosum (CC) of the ALS group, extending rostrally to the motor cortices. Regional analysis of radial diffusivity increase (a DTI measure thought to reflect Wallerian degeneration), closely matched the FA regional changes. FA changes detected in the more caudal corticospinal tracts (CST) were less marked than those seen rostrally. All the FA changes (including the CC) were strikingly consistent with historical postmortem neuropathological findings in ALS patients (1).

CC changes were independent of UMN involvement. There was a limited correlation with disability. A separate whole-brain analysis by clinical UMN involvement delineated just the CSTs. GM changes were widespread and consistent with areas of WM involvement. Post hoc discriminant analysis using the combination of FA, RD and GM values separated the two groups with 92% sensitivity and 88% specificity.

Discussion: CC involvement is a consistent feature of ALS across a range of phenotypes, independent of clinical UMN involvement, and may reflect early inter-hemispheric spread of pathology. Transcranial magnetic stimulation (TMS) studies through the CC have previously been noted to be abnormal in ALS.

The notable delineation of the CST in whole-brain analysis of FA in relation to clinical UMN involvement provided further validation of DTI as a sensitive tool for detecting neuronal pathology in ALS.

Conclusions: Our findings support an inherent cerebrally-driven pathological process, rather than simple anterior horn cell ‘dying back’ in ALS. CC changes may have particular potential as an early biomarker in those identified in the future to be ‘at risk’, possibly through a combination of DTI and TMS.

Reference:

• Smith MC. J Neurol Neurosurg Psychiatry 1960;23:269–82.
   PubMed Web of Science ®Google Scholar

P201 FINGERPRINT CHARACTERISTICS OF RARE MOTOR NEURON DISORDERS BY APPLICATION OF DIFFUSION TENSOR IMAGING METHODS

UNRATH A¹, MUELLER H-P¹, SPERFELD A-D², LUDOLPH AC¹, KASSUBEK J¹

¹University of Ulm, Department of Neurology, Ulm, Germany, ²HELIOS Clinics Bad Saarow, Department of Neurology, Bad Saarow, Germany

E-mail address for correspondence: [email protected]

Keywords: DTI, pathoanatomy, biomarker

Background and objectives: Motor neuron disorders (MND) are clinically defined by the predominance of upper or lower motor neuron involvement and the course of the disease. Magnetic resonance imaging (MRI) basically serves as a diagnostic tool to exclude other pathologies, but gradually novel approaches based on automated and computer-assisted techniques, such as diffusion tensor imaging (DTI) methods help to identify the pathophysiological processes of these disorders within the cerebral white matter (WM) in vivo. This study was designed to investigate different rare MND in order to define pathoanatomical characteristics.

Methods: Five groups of rare motor neuron disorders were included in this study, ie primary lateral sclerosis (PLS, N=25), pure and complicated hereditary spastic paraparesis (pHSP, N=24 and cHSP, N=14, respectively), X-linked spinobulbar muscular atrophy (X-SBMA, N=20) and spinal muscular atrophy (SMA, N=21). Whole-brain based DTI analysis methods were applied to the patient groups in comparison with matched controls. All data analyses were performed by use of the DTI software TIFT (Tensor Imaging and Fiber Tracking).

Results: WM analysis revealed widespread affectations and characteristic patterns of alterations within the supratentorial motor system in all patient groups with varying predominance according to the clinical focus, noteworthy also in disorders with a clinically defined isolated involvement of the lower motor neuron. Further reductions of the fractional anisotropy as a marker of WM integrity were observed within distinct regions of the corpus callosum in the pHSP and particularly cHSP group as well as in PLS patients, additionally in non-motor regions in projection to the limbic system in X-SBMA and both HSP groups. The most widespread extra-motor WM affectations were observed in the cHSP group.

Discussion and conclusion: In summary, DTI was able to delineate a characteristic WM pathoanatomy in motor and extra-motor brain areas for different MND via whole brain-based fractional anisotropy assessment which provides a fingerprint identification of these disorders. Future developments should aim at multiparametric imaging analyses to explore biomarker information.

P202 RESEARCH ON THE CLINICAL FEATURES, MAGNETIC RESONANCE IMAGING AND ELECTROMYOGRAPHY OF 28 CASES DIAGNOSED WITH HIRAYAMA DISEASE

ZENG Y, HU X-H, HUANG R, FANG D-F, GUO X-Y, SHANG H-F

Department of Neurology, West China Hospital of Sichuan University, Chengdu, Sichun, China

E-mail address for correspondence: [email protected]

Keywords: MRI, electromyography, Hirayama Disease

Objective: To study the clinical, magnetic resonance imaging (MRI) and electromyography (EMG) features in Hirayama Disease (HD).

Methods: Twenty-eight patients with HD were included in the study. Clinical information, results of cervical MRI in a neutral and a fully flexed position, and results of EMG of ulnar nerve and median nerve in a neutral and a fully flexed position were analyzed.

Results: The 28 cases consisted of 27 males and 1 female and the mean onset age was 16.9±1.56 years. The mean delay between age at onset and age at diagnosis was 1.80±1.00 years (except for 1 case with delay of 18 years). The clinical symptoms and signs of these patients were not progressed with follow-up of 1–3 years. Twenty-five patients had symptoms and signs on one side only (left to right ratio: 8/17). Both sides were affected in 3 patients. Cervical spinal cord MRI of all patients showed typical HD MRI features. EMG tests revealed abnormalities of neurogenic changes in all clinically affected upper limbs in all patients. Clinically unaffected upper limbs of 11 patients (44%) who had unilateral clinically affected upper limbs were also found to have abnormalities by EMG test. The frequency of F wave for ulnar and median nerves of clinically affected limbs was significantly lower than that of unaffected upper limbs (P<0.05) whenever the neck was in a neutral or flexed position. The frequency of F wave of ulnar and median nerves when the neck was kept in a neutral position was significantly lower than that in a flexed position whenever testing the clinically affected or unaffected upper limbs (P<0.05).There was no significant difference in the frequency of F wave of the median nerve in clinically unaffected upper limbs between neutral and flexed neck positions (P=0.144).The amplitude of F wave of the median nerve in clinically affected limbs when the neck was kept in neutral position was significantly higher than that in a flexed position (P=0.048).

Conclusion: Adolescents presenting unilateral limb weakness and/or atrophy should be considered with Hirayama disease, and confirmed by cervical spinal MR imaging in a neutral and a fully flexed position. EMG test is sensitive to find abnormality in the clinically unaffected limb. The frequency and amplitude of F wave may be a useful index to evaluate the severity and progression of Hirayama Disease.

P203 CHANGES IN ELECTROPHYSIOLOGICAL MARKERS OF COGNITION IN ALS: PRELIMINARY RESULTS OF A HIGH-RESOLUTION EEG STUDY

STUKOVNIK V¹, DREO J¹, BON J^2,4, PODNAR S¹, ZIDAR J¹, REPOVš G³

¹Institute of Clinical Neurophysiology, University Medical Centre Ljubljana, Ljubljana, Slovenia, ²Psychiatric Hospital Begunje, Begunje, Slovenia, ³Department of Psychology, Faculty of Arts, University of Ljubljana, Ljubljana, Slovenia, ⁴Division of Neurology, University Medical Centre Ljubljana, Ljubljana, Slovenia

E-mail address for correspondence: [email protected]

Keywords: high-resolution EEG, event related potentials, cognition

Objective: Amyotrophic lateral sclerosis (ALS), traditionally considered exclusively as a motor system disorder, is increasingly recognized as a multi-system disease, affecting also non-motor areas, with results ranging form subtle behavioral and executive dysfunctions to, less frequently, a clear fronto-temporal dementia. An increasing need for reliable assessment of cognitive impairments in ALS is not easily met by regular psychometric methods due to their dependence on patients’ unimpaired motor function. One of the recently considered motor-free alternatives in the assessment of cognitive impairments is the observation of changes in neuro-correlates of cognitive processes such as late event-related potentials (ERPs). While existing ERP studies of ALS have indeed identified changes in cognition-related ERPs, they have so far failed to identify sensitive markers of cognitive dysfunction, leading the authors to question the utility of ERP measurements in clinical ALS evaluation. Most of the studies were however limited to single or multiple channel waveform analysis and have not made full use of the topographical information provided by dense-array EEG recording. The aim of this study was to explore possible electrophysiological markers of cognitive change in non-demented patients with early-stage sporadic ALS using topographical ERP analysis of dense-array EEG recordings.

Methods: We recruited 12 patients with sporadic ALS (9 with limb-onset, 3 with bulbar-onset), all fulfilling the criteria of a ‘probable or definite’ ALS according to international classification of El Escorial – Revised and 11 healthy control subjects. We used a classical visual two-stimulus ‘oddball’ counting paradigm to evoke P3 ERP. Subjects’ EEG was recorded with a 128-channel EEG (Brain Products actiCAP) recording system according to the International 10-5 System. Each participant performed at least 4 blocks of trials comprised of 100 stimuli each, counting mentally the number of targets and reporting it at the end of each block. The Spherical Spline Laplacian (SSL) method was used to estimate average cortical surface potentials in target condition for each subject. A template response was then computed based on the control group grand-average, and both groups were compared on how well they matched the template in ERP amplitude, topography and time-course.

Results: Comparison of patient and control groups showed marked differences in all three ERP analysis domains: amplitude, topography and latency. Using binary logistic regression, topography and time-course data provided near perfect prediction of group membership.

Conclusion: Though preliminary, our findings indicate the presence of robust differences in late cognition-related ERPs in non-demented, sporadic ALS patients. Not relying on intact motor function, cognitive ERPs may provide a fruitful alternative to classical psycho-metric methods for testing cognition in ALS patients. Current results indicate that it might be possible to further develop this ERP method for the purposes of diagnosing and monitoring the progression of the disease.

P204 MOVEMENT-RELATED CORTICAL POTENTIALS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

BIZOVICAR N, ZIDAR I, KORITNIK B, ZIDAR J

Institute of Clinical Neurophysiology, University Medical Centre Ljubljana, Ljubljana, Slovenia

E-mail address for correspondence: [email protected]

Keywords: movement related cortical potential, functional tests, respiration

Background: Respiratory failure is the main cause of death in patients with ALS. It is not known whether upper motor neuron loss contributes to it. Sniff nasal inspiratory pressure is a reliable marker of respiratory insufficiency in ALS. Therefore we have chosen sniffing to study cortical neural correlates of voluntary breathing. Each voluntary movement is preceded by a movement-related cortical potential (MRCP) that can be recorded from the scalp. It reflects the activity of the primary and secondary motor cortical areas. When associated with sniffing, it is called sniffing-related cortical potential (SRCP).

Objectives: Our aim was to study MRCPs related to finger flexion and sniffing as possible neural markers of the upper motor neurone dysfunction of hand and respiratory muscles. We searched for correlations between the SRCP amplitudes and the results of tests that measure respiratory function and also between the MRCP amplitudes obtained by finger flexion and the hand function tests.

Methods: Fourteen ALS patients (aged 42–77 years) and 15 healthy volunteers (aged 45–71 years) were studied. None of them had symptoms or clinical signs of respiratory insufficiency. They were assessed for their hand dexterity and strength, respiratory function, speech capacity, and upper motor neuron burden, Ashworth, Norris, and ALS functional rating scales. Electromyographic parameters used were neurophysiological index and the diaphragm CMAPs. Subjects performed self-paced sniffing every 5–10s with 20% of maximal sniff inspiratory pressure and the right finger flexion every 5–10s with 20% of maximal finger flexion pressure. The nasal pressure and the pressure produced by finger flexion were used as signals for back averaging of the EEG (10–10 system, 32 electrodes). The SRCP and MRCP amplitudes detected by electrodes Cz, C3, C4, FC5 and FC6 were measured.

Results: No significant differences of SRCP and MRCP amplitudes were found between the patients and healthy control subjects. In patients, there was a significant positive correlation between the SRCP amplitudes and scores of the Ashworth and bulbar part of the upper motor neuron burden scales, and oxygen saturation. Significant negative correlation was found between the SRCP amplitude and scores of the Norris scale. Significant negative correlation was also found between the MRCP amplitude and scores of the Ashworth scale and the time needed to perform the nine-hole peg test. There was a positive correlation between the MRCP amplitude and the results of the Tapping Board test.

Conclusions: Patients as a group did not have smaller amplitudes of the cortical potentials. Amplitudes of SRCPs were mostly not related to respiratory function tests. Higher amplitudes of finger-flexion MRCPs were related to better hand function. This finding maybe offers a possibility of using the MRCPs as biomarkers of disease severity.

P205 UPPER TRAPEZIUS ELECTROMYOGRAPHY AIDS EARLY DIAGNOSIS OF BULBAR INVOLVEMENT IN AMYOTROPHIC LATERAL SCLEROSIS

XU Y, ZHENG J, ZHANG S, ZHANG J, FAN D

Peking University Third Hospital, Beijing, China

E-mail address for correspondence: [email protected]

Keywords: electromyography, trapezius, bulbar involvement

Background and objectives: Electromyography (EMG), particularly tongue or sternocleidomastoid EMG, aids in amyotrophic lateral sclerosis (ALS) diagnosis and can be used to identify lower motor neuron lesions in the bulbar region. Abnormal trapezius EMG was recently shown to be useful in ALS diagnosis. Here we investigated the role of upper trapezius EMG in assessing bulbar involvement in ALS.

Methods: A standard EMG was recorded from the upper trapezius in ALS patients, cervical spondylotic myelopathy (CSM) patients, and normal controls. Forty-three CSM patients were examined pre-operation and three months post-operation.

Results: Greater spontaneous activity levels were seen in the upper trapezius EMG of ALS patients with disease durations of ≤ 8 months (70%) than in patients with durations of > 8 months (40%). Significant differences in the motor unit action potential parameters were noted between ALS patients and normal controls or CSM patients. Fewer spontaneous activities were detected post-operation in CSM patients. No differences in neurogenic EMG changes were observed between the trapezius and sternocleidomastoid in ALS patients.

Conclusions: Upper trapezius EMGs may provide valuable information for assessing the clinical and subclinical involvement of bulbar lower motor neurons in ALS patients, particularly at early disease stages.

P206 STERNOCLEIDOMASTOID AND SCALENUS ANTERIOR MUSCLES INVESTIGATION IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS: AN ELECTROMYOGRAPHIC STUDY

DE CARVALHO M^1,2, PINTO S², SWASH M^3,2

¹Department of Neurosciences, Hospital de Santa Maria, Lisbon, Portugal, ²Neuromuscular Unit, Instituto de Medicina Molecular, Lisbon, Portugal, ³Department of Neurology, Royal London Hospital, Queen Mary School of Medicine, University of London, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: electromyography, diagnosis, monitoring

Background: To confirm diagnosis of ALS on electromyography (EMG) it is important to disclose abnormal findings in cranial innervated muscles. It is surprising that in patients with weak neck flexion the Sternocleidomastoid (SCM) muscle is frequently normal on EMG.

Objectives: We aimed to test SCM and scalenus anterior (SA) in ALS to test sensitivity of EMG applying different new techniques.

Methods: SM and SA were tested in 49 and 38 controls, respectively. SA needle placement was confirmed by ultrasound in 2 controls. In this population interferential pattern analysis of the EMG signal was performed, obtaining: number of turns (change in the motor unit potential polarity in the same phase), mean amplitude (amplitude of turns), turns/amplitude ratio, number of short-segments (number of intervals between successive turns); activity (percentage of time with EMG activity) and enveloped amplitude (amplitude without outliers). In addition in 25 (SCM) and 20 controls (SA) the new method of peak-ratio (PR) was calculated (peak-ratio and number of short-segments). The same approach was performed in 60 ALS patients (PR in a subset of 37 patients). Both groups were matched for sex and age. All ALS patients had probable or definite disease, with ALS-FRS > 25 and neck flexor strength > 3 (MRC) and 27 had bulbar-onset disease. A P value < 0.01 was taken as significant.

Results: Comparing controls vs ALS, for both SCM and SA, number of turns, turns/amplitude, activity, number of short-segments and PR showed differences. In SCM, 42% of ALS patients had at least one abnormal turn-amplitude parameter, and in SA 67% showed this abnormality (P>0.01). For PR, this number was 27% and 72% for SCM and SA, respectively (P<0.001). The most abnormal turn-amplitude parameter was number of short-segments for both SCM (25%) and SA (53%). In SCM and SA we did not find a difference in the frequency of abnormalities between bulbar and spinal-onset patients.

Discussion and conclusions: Both turn-amplitude analysis and PR are easy to perform in neck muscles and disclose a large number of abnormalities in early-affected ALS patients. The sensitivity is similar between both methods. The SA shows more changes than the SCM muscle, supporting the observation of normal SCM on EMG in patients with weak neck flexion. Neither muscle is particularly more sensitive in bulbar-onset patients. SA is useful in the EMG assessment of a diagnosis of ALS.

P207 METABOLIC DYSREGULATION OF LIPID, URATE AND IRON IN SERA OF PATIENTS WITH SPORADIC AMYOTROPHIC LATERAL SCLEROSIS: CORRELATION BETWEEN SEROLOGICAL CHANGES AND DISEASE PROGRESSION

IKEDA K, YOSHII Y, KAWABE K, ISHIKAWA Y, IWAMOTO K, IWASAKI Y

Toho University Omori Medical Center, Tokyo, Japan

E-mail address for correspondence: [email protected]

Keywords: lipids, urate, ferritin

Background: Several pathogenetic and prognostic factors are speculated in familial and sporadic forms of amyotrophic lateral sclerosis (ALS). Previous reports suggested distinct profiles of serum lipid, urate and ferritin levels in ALS patients. Dyslipidemia and hyperuricemia are protective markers and suggest a favorable prognosis in ALS patients.

Objectives: The purpose of this study is to examine serological changes of lipid, urate, and iron metabolites in Japanese patients with sporadic ALS. We also analyzed whether those serum levels were linked to disease course.

Methods: Seventy-nine patients with definite or probable ALS fulfilling the El Escorial revised criteria were analyzed for age, sex, limbs or bulbar onset, disease duration, ALS Functional Rating Scale (FRS), body mass index (BMI) and forced vital capacity (FVC), and serum levels of the following 9 items; total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglyceride, urate, iron, transferrin, ferritin and creatinine. Those variables were measured twice at the initial examination and one year later. All patients had neither family history of ALS nor mutations of superoxide dismutase (SOD) 1. ALS patients who had hypertension, renal dysfunction, gout, chronic inflammatory diseases, a percutaneous enterogastrostomy, iron medication, and UA- and lipid-lowering prescription were excluded from this study. Serological variables were compared between ALS patients and 80 healthy controls matotal cholesterolhed by age, sex and BMI.

Results: As compared to controls, serum urate and creatinine levels were decreased significantly in male and female patients with ALS. Serum ferritin levels were increased significantly in men and women with ALS. A significant reduction of serum high density lipoprotein cholesterol levels, and significant elevations of serum total cholesterol and triglyceride levels were found in female ALS patients. No statistical differences of total cholesterol, low density lipoprotein, high density lipoprotein and triglyceride existed between sera of male ALS patients and controls. Serum iron and transferrin levels did not differ between male and female ALS and controls. Impairment of FRS and FVC were delayed significantly in ALS patients with serum total cholesterol levels ≥ 200 mg/dL or low density lipoprotein ≥ 130 mg/dL at baseline. Annual decline rate of FRS was correlated with decline rate of serum creatinine levels, BMI and FVC. Baseline data of high density lipoprotein cholesterol, triglyceride, urate, iron, transferrin and ferritin did not influence decline rate of FRS.

Discussion and conclusions: The present study indicated significant decrease of urate levels and increase of ferritin levels in sera of ALS patients. Those serum levels were not associated with disease progression, including FRS, FVC and BMI. Higher serum total cholesterol and low density lipoprotein-C levels suggested favourable prognosis of ALS patients. Metabolic alternation of lipid, urate and ferritin could contribute to the pathogenesis and the progression of patients with sporadic ALS without SOD1 mutations.

P208 NO CORRELATION OF GANGLIOSIDE ANTIBODIES WITH DISEASE PHENOTYPE AND SURVIVAL IN AMYOTROPHIC LATERAL SCLEROSIS

KOLLEWE K, SINZENICH T, MOHAMMADI B, DENGLER R, WURSTER U, PETRI S

Medical School Hannover, Hannover, Germany

E-mail address for correspondence: [email protected]

Keywords: ganglioside antibodies, phenotype

Introduction: Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder with typical onset in the fifth- sixth decade of life. The earlier hypothesis of an autoimmune origin of ALS receives less attention today, but immunological phenomena still seem to be involved and mechanisms such as protective autoimmunity may be important. Antibodies against a variety of gangliosides also occur in ALS, but widely differing frequencies and titers have been reported. We investigated the presence of IgG and IgM antibodies in ALS patients.

Methods: IgG and IgM antibodies to the six gangliosides asialo-GM1 (GA1), GM1, GM2, GD1a, GD1b, GQ1b were determined by GanglioCombi ELISA in sera of 84 ALS patients with a mean age of 58. Results were expressed as a % ratio of a highly positive GM1 control and categorized as negative (<30%), borderline (30–50%), moderately (50–100%) and strongly positive (>100%). The values obtained from 220 Swiss blood donors (mean age: 52) served as a reference group.

Results: Twenty-two (26.2%) out of 84 ALS-patients showed elevated ganglioside antibodies: Taking all subspecific antibodies together, IgG antibodies were found in 9/84 (10.7%) and IgM in 15/84 (17.9%) patients. As 2 patients exhibited both isotypes, the combined frequency is 22/84 (26.2%). Three simultaneous antibodies occurred in 1 patient each for IgG (GA1, GD1a, GQ1b) and IgM (GM1, GD1a, GD1b). However, the frequencies for the individual antibodies were rather low, with maximal 7.1% (6/84) for IgG GD1a and 8.3% (7/84) for IgM GA1. Strongly positive values were found in 4 patients, 2 for IgG (GA1, GD1a) and 2 for IgM (GA1 and GM1). There was no statistically significant difference to the collective of normal blood donors.

Conclusions: Several studies with controversial results have assessed a potential association between elevated ganglioside-antibody-titres in ALS patients and specific disease phenotypes. Whilst in the existing literature mainly GM1- ganglioside antibodies were analysed, we used a novel assay to detect six subspecific IgM and IgG antibodies. Even with this more thorough approach, ganglioside antibody frequencies and patterns in our ALS cohort closely resemble the values observed in healthy controls, and the presence of ganglioside antibody was not correlated with age, gender, ALS phenotype or survival.

P209 ELIMINATING RATE IN LACTATE STRESS TEST: A POTENTIAL PREDICTOR FOR ALS SURVIVAL

ZHANG Y, FAN D

Peking University Third Hospital, Beijing, China

E-mail address for correspondence: [email protected]

Keywords: lactate stress test, eliminating rate, progression

Objectives: Mitochondrial dysfunction plays an important role in the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we aimed to prove mitochondrial dysfunction in ALS by lactate stress test, and explore a possible association between lactate levels and clinical characteristics of ALS.

Methods: We carried out a cross-sectional study in 116 consecutive ALS patients. A standard procedure was performed: ALS patient rested completely for at least 15 minutes. After rest immediately before the test, lactate was determined for the first time (lactate before). Afterwards the patient was told to cycle with continuously close to uniform effort for 15 minutes, rotation speed 20–40 r/min, by a paddle-rate-independent electronic bicycle ergometer. Immediately after exercise a blood sample was acquired for lactate test (Lactate 0). Finally, after a rest for 15 minutes lactate was determined again (lactate 15). Plasma lactate concentrations were determined by high-performance liquid chromatography with fluorescence detection, whereas pyruvate at every time-point was determined also according to established guidelines. We calculated five deuterogenic values to seek an appropriate index, which are: (1)Eliminating rate (ER)=(lactate15- Lactate0)/15; (2)(Lactate15/Lactate before) ratio; (3)(Lactate0/Lactate before) ratio; (4)(Lactate 0/Pyruvate Before) ratio; (5)(Lactate before/Pyruvate before) ratio.

Results: We found a trend for patients with rapid slope of ALSFRS-r (>20 U/year) to have a slowest ER (median −4.67*10⁻³mmol/L/min, Coefficient of variation, 590.15%), shortest duration (0.63±0.28 years) and worst ALSFRS-r (32.59±4.93). The patients with moderate slope of ALSFRS-r (10–20 U/year) have a moderate ER (median −11.33×10⁻³mmol/L/min, Coefficient of variation, 309.89%), duration (1.16±0.45 years) and ALSFRS-r (34.83±6.11). The slower progression (<10 U/year) patients have a rapid ER (median: −12.00×10⁻³mmol/L/min, coefficient of variation: 143.08%), longer duration (median: 3 years, coefficient of variation: 193.33%) and good ALSFRS-r value (39.58±9.44). Furthermore, we found advanced phase ALS patients (defined as definite and probable ALS) also have slower ER (quariles −17.33, −5.67, 4) and worse ALSFRS-r (34.88±9.27), while early phase patients (defined as laboratory supported probable and possible ALS) have more rapid ER (quariles −25.17, −11.33, −3.50) and better ALSFRS-r (39.28±7.59). All the differences are statistically significant. Multiple linear regression revealed a strong direct association between ER, ALSFRS-r slope (standard Beta=0.33, P=0.007) and FVC (standard Beta= −0.458, P=0.006, adjusted for ALSFRS-r, course and onset region).

Conclusions: This study was a cross-sectional design of clinical variables and laboratory numeric values, which prevented the assessment of a temporal relationship between decreased ER and motor decay. Our results suggested that slower ER may be linked to faster progression of the disease and has potential to be a biomarker for ALS survival.

P210 PATHOLOGICAL CHANGES IN THE LATERAL CORTICOSPINAL TRACT IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS ARE ASSOCIATED WITH ALTERED NEUREGULIN EXPRESSION

SONG F, CHIANG P, LOEB J

Hiller ALS Clinic and Research Center, Department of Neurology, Center of Molecular Medicine and Genetics, Wayne State University, Detroit, MI, United States

E-mail address for correspondence: [email protected]

Keywords: neuregulin, lateral corticospinal tract, biomarker

Background: Amyotrophic lateral sclerosis (ALS) is poorly understood at the molecular level and there are as yet no effective therapeutics to stop the insidious progression that involves both the upper and lower motor systems. ALS mainly affects lower motor neurons in the spinal cord that are functionally related to upper motor neurons in the cortex through monosynaptic connections (1). Surprisingly even less is known about the cause of pathological progression within the lateral corticospinal tract (LCST) that connects the upper and lower motor systems. These monosynaptic interactions between upper and lower motor neurons are not affected in the most commonly studied mouse and rat models (2), leaving only studies on postmortem human tissues to begin to understand this region of progressive degeneration in ALS.

Objectives: Here we have set out to identify biomarkers as well as potential drug targets from postmortem human ALS patients with special attention to the pathological changes in the LCST.

Methods: Pathological changes were measured for motor neuron number, axon loss, demyelination, gliosis and microglial activation in both patients with ALS and controls. Gene expression and protein levels were determined by quantitative PCR for a number of genes implicated in the disease process as well as the NRG1 gene isoforms that have important roles in nervous system development.

Results: The spinal cord and brain of patients with ALS showed varying degrees of neuronal loss and activation of microglial and astrocytes both within the ventral horn and LCST. While there was no consistent change in SOD1, TDP43, and GLT1 (genes implicated in pathogenesis), there was a significant downregulation of NRG1 gene expression (a neuronally-expressed gliotrophic gene that supports axoglial interactions) in the spinal cord but slight upregulation in the brain. Up-regulation of type III NRG1 protein expression was observed both in the brain and the LCST and was associated with axonal loss and demyelination in the LCST and with microglial activation/infiltration. The down-regulation of type III NRG1 protein in spinal motor neurons is also microglial activation in the ventral grey matter.

Conclusions: Type III NRG1, an important glial regulatory factor produced by neuronal axons, is dysregulated in both upper and lower motor neurons in patients with ALS in both the ventral horn and LCST.

Discussion: Altered expression of this important growth and differentiation factor could result in altered cell-cell signal patterns in ALS that could both lead to disease progression as well as serve as a biomarker for neuronal loss and pathological changes within the LCST.

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P211 A HIGH THROUGHPUT IMAGE ANALYSIS STRATEGY FOR ANALYSIS OF IN SITU HYBRIDIZATION TRANSCRIPT PATTERNS IN THE SOD1_G93A MOUSE MODEL OF ALS

LINCECUM J¹, WANG M¹, PUCHALSKI R¹, SANCHEZ R¹, CARRION I¹, VIEIRA F¹, PERRIN S¹

¹ALS TDI, Cambridge, MA, United States, ²Allen Institute for Brain Science, Seattle, WA, United States

E-mail address for correspondence: [email protected]

Keywords: ISH, SOD1_G93A, algorithm

The recent acceleration of the pace of high throughput genomics and the generation of complex gene networks has underscored the need for technologies capable of capturing and quantifying gene expression patterns in tissues. The traditional method for visualizing patterns of gene expression is in situ hybridization (ISH). ISH originally employed radioactive probes, manual hybridization to processed tissue sections, and arduous autoradiography to see patterns of gene expression. This radioactive method has since been improved using ‘cold’ techniques based on hapten labeled riboprobes and immunohistochemical detection of the signal. Optimized methods for automation of tissue and slide processing with high throughput imaging have made possible large scale projects such as the Allen Institute for Brain Science's Mouse Brain Atlas and Mouse Spinal Cord Atlas projects (www.brain-map.org). Working in collaboration with the Allen Institute we have generated a database of several thousand transcript patterns covering the presymptomatic and early symptomatic spinal cord of SOD1_G93A mouse model of ALS.

Here we report on our efforts to implement an automated solution to the image analysis of these patterns of gene expression. Using a software package, CellProfiler 2.0 (1) we have developed an image analysis pipeline capable of quantifying patterns of transcript expression during disease progression in the SOD1_G93A spinal cord. The algorithm has been successfully applied to a panel of neuronal and inflammatory markers, such as GFAP and CD68, and shown to be robust and reproducible. In conclusion, automated image analysis of complex patterns of gene expression makes possible development of biomarker panels of cell state applicable to pre-clinical pharmacodynamic screening in this mouse model of ALS.

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P212 BIOMARKERS IN A TRANSGENIC MOUSE MODEL OF NEURODEGENERATION

CALVO A¹, TORRE-MERINO P², MANZANO R¹, OLIVáN S¹, MUñOZ JM¹, ZARAGOZA P¹, GARCíA-REDONDO A², OSTA R¹

¹LAGENBIO-I3A, Aragonese Institute of Health Sciences (IACS), Faculty of Veterinary Science, University of Zaragoza, Miguel Servet, Zaragoza, Spain, ²Servicio de Neurología - Unidad de ELA, CIBERER U-723, Instituto de Investigación Sanitaria Hospital 12 de Octubre, Madrid, Spain

E-mail address for correspondence: [email protected]

Keywords: biomarker, muscle biopsy, muscle atrophy

Background: Amyotrophic Lateral Sclerosis (ALS), a progressive motor neuron disease, is one of the most common neurodegenerative disorders. ALS causes muscle atrophy together with a degeneration of motor neurons leading to death within 3 to 5 years after onset. Many studies have been carried out to detect molecular markers for diagnosis of ALS in different tissues like blood or cerebrospinal fluid. However, the molecular targets that prompt the neurodegenerative process in ALS remain unknown.

Objectives: Our main goal is to study, throughout disease progression, the levels of expression of groups of genes directly related to the atrophy and regeneration of skeletal muscle, one of the tissues most affected by the disease, in order to find molecular biomarkers for ALS.

Methods: Inbred B6SJL SOD1^G93A mice were used as they have been established as a suitable ALS disease model. Muscle biopsies were carried out in males and females mice at the asymptomatic, symptomatic and terminal stages. Biopsy samples were processed for total RNA extraction. Gene expression variations in all samples were assayed by real-time PCR. Genes related to muscle damage; muscle differentiation and regeneration; maintenance of muscle integrity and muscle reinnervation; calcium homeostasis; glucose homeostasis and oxidative stress were studied. Statistical analysis was carried out to find correlation between gene expression levels throughout disease progression and longevity.

Results: Genes involved in the maintenance of muscle integrity and in the inhibition of muscle reinnervation correlated with longevity in both males and females. Furthermore, genes involved in muscle damage, muscle differentiation and regeneration, glucose metabolism and calcium homeostasis were correlated to longevity only in females.

Discussion and conclusions: The different gene expression profile and correlations found in males and females suggest that the skeletal muscle behaves in a different way in both sexes under neurodegenerative conditions. Female transgenic mice seem to compensate for the activation of degeneration signals in skeletal muscle more efficiently than males, delaying muscle differentiation and buffering deregulation of calcium and glucose homeostasis. These results could shed light to find new biomarkers in different tissues in SOD1^G93A mice and in a later step to translate the study of these biomarkers to human samples, so as to finally reach a more accurate knowledge of the disease.

Acknowledgements: This work was supported by the grants: Fondo de Investigación Sanitaria-Instituto de Salud Carlos III (PI071133) and the Project “Tú eliges: tú decides” of Caja de Ahorros de Navarra in Spain.

P213 DYNAMIC IN VIVO OPTICAL IMAGING AND MRI FOR TRACKING NANOPARTICLES AND EVALUATING DISEASE PROGRESSION IN SOD1(G93A) MICE

SHARP P, BERWICK J, KENNERLEY A, WANG L, MADSEN J, ARMES S, MEAD R, SHAW P, AZZOUZ M, BATTAGLIA G

The University of Sheffield, Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: nanoparticles, MRI, optical imaging

Background: A major obstacle in the treatment of neurological disorders is the blood-brain-barrier (BBB), which limits the entry of many potentially therapeutic agents into the central nervous system (CNS). Advances in nanotechnology have led to the development of biocompatible nanoparticles, which can act as nanometer-sized vectors for therapeutic agents. One promising class of nanoparticle, known as polymersomes, are made of amphiphilic block copolymers that self-assemble to form polymeric vesicles. We have previously demonstrated efficient cytosolic delivery of active agents using polymer vesicles in numerous cell lines. Our aim now is to examine their application in vivo to effectively deliver neuroprotective agents into the CNS. To enhance BBB translocation, polymeric vesicles will be functionalized (ligand-decorated) to target receptor-mediated transcytosis at the BBB. Optimal formulations identified will be used to enhance delivery of therapeutic agents in SOD1(G93A) mice.

Objectives: To evaluate the potential of numerous polymersome formulations, we aim to develop a quantitative in vivo optical imaging technique to characterize the efficiency in which fluorescently labeled polymersomes can traverse the BBB. Furthermore, in preparation for therapeutic testing in SOD1(G93A) mice, we also aim to establish the use of high-field MRI to precisely measure key pathological markers of disease.

Methods: Cerebral vasculature was visualized through a cranial window of terminally anaesthetized rats. Rhodmine labeled polymersomes were administered intravenously and fluorescent signal was measured in the vascular and extravascular compartments. For the longitudinal MRI study, SOD1(G93A) transgenic mice (n = 5) and non-transgenic littermates (n = 5) were scanned every 30 days throughout their life span.

Results: Dynamic image analysis of cerebral vessels in vivo showed that the fluorescent signal generated by rhodamine labeled polymersomes was readily detectable and stable. In contrast, signal generated by free rhodamine was highly transient, demonstrating the stability of polymersomes in circulation. The polymersomes examined were not functionalized, so no increase in signal in extravascular regions was detected. MRI scans showed that hindlimb and pelvic muscle volumes of SOD1(G93A) transgenic mice were significantly smaller than controls as early as 30 days of age, which is almost 50 days prior to the onset of overt behavioural signs. Subsequent analysis showed a considerable difference between transgenic and control mice at 60, 90, and 120 days of age. In addition, clearly defined lesions were also detected in the brainstem of transgenic mice at 90 and 120 days of age.

Discussion and conclusions: We report here a novel optical imaging based methodology for sensitive monitoring of fluorescently labeled nanoparticles in vivo. This approach will be invaluable for assessing BBB penetration of nanoparticle-based platforms. We also demonstrate that MRI is a powerful imaging tool for tracking pathological changes in SOD1(G93A) mice, which will be applied in future preclinical efficacy studies.

P214 PROGRESSIVE CHANGES IN SPINAL MOTOR SYSTEM IN AN ADULT MOUSE MODEL OF AMYOTROPHIC LATERAL SCLEROSIS

JIANG M, SCHUSTER J, FU R, SIDDIQUE T, HECKMAN CJ

Northwestern University, Chicago, Illoise, United States

E-mail address for correspondence: [email protected]

Keywords: root recording, interneurons, bursts

Background: Amyotrophic lateral sclerosis (ALS) is a late onset motor disease characterized by progressive degeneration of motoneurons. Excitotoxicity is one of several mechanisms being investigated for motoneuron degeneration in ALS. It is generally considered that the excitotoxicity arises from excessive Ca²⁺ entry into motoneurons and the poor motoneuronal Ca²⁺ buffering. The excessive Ca²⁺ entry may occur when excitatory synaptic inputs or voltage-sensitive Ca²⁺ channels become more active, resulting in increased opening of either Ca²⁺ permeable AMPA and NMDA receptors or voltage-sensitive Ca²⁺ channels. On the other hand, decrease in inhibitory synaptic transmission may disinhibit motoneurons and cause increase of Ca²⁺ entry. Thus, identification of detailed excitotoxic source is critical for its pathogenesis and therapy.

Objectives: The objective of this investigation was to evaluate the peripheral monosynaptic inputs and spinal pre-motor network that may contribute to the excitotoxicity of spinal motoneurons of mSOD1 mice.

Methods: Under urethane anesthesia, spinal cords containing sacral 1–3 segments (S1-3) and their ventral and dorsal roots were taken from mSOD1^G93A mice and their nontransgenic littermates (NG) at three age groups (P50, P90 and > P120), and mounted in a recording chamber superfused with artificial brain-spinal fluid. The dorsal roots and distal ends of ventral roots were placed on stimulating electrodes while the proximal ventral roots were placed on recording electrodes. Chemicals were added to spinal cord through bath application. The evoked root responses were compared between mSOD1 and NG mice. To test spinal interneurons, strychnine, the glycine receptor antagonist, and picrotoxin, the GABAa receptor antagonist, were applied to generate synchronized bursts. Short train stimulation (five pulses) was also delivered to the dorsal root to determine the role of inhibitory synapses in ALS.

Results: The ventral root responses evoked by both peripheral and ventral root stimulation progressively decreased with age, making the peripheral monosynaptic input unlikely as an excitotoxic source. After symptom onset, a higher percentage of mSOD1 mice exhibited bursting with more sub-bursts and increased randomness. In mSOD1 mice with clear muscle tremor, the ventral roots exhibited spontaneous synchronized bursts, which were highly sensitive to NMDA receptor blockade. It was also demonstrated that inhibitory synapses participate in short-term depression (STD) at peripheral-motoneuron synapses.

Discussion: Although the induced and spontaneous bursts in mSOD1 mice indicate a role of interneurons in ALS pathogenesis, the involvement of receptors requires further investigation. By testing STD, this continuing study examines the functional status of GABAa/glycine receptors between mSOD1 and NG mice.

Conclusion: The data suggest progressive increase in excitability of spinal interneuons in mSOD1 mice that may lead to an excitotoxic effect on motoneruons.

P215 THE EFFECT OF THE ELECTRICAL AND MORPHOLOGICAL ALTERATIONS IN MUTANT SOD1 MOTONEURONS ON THE DENDRITIC PROCESSING OF SYNAPTIC INPUTS

Elbasiouny S¹, Durand J², Heckman CJ¹

¹Northwestern University, Chicago, IL, United States, ²CNRS, Université de la Méditerranée, Marseille, France

E-mail address for correspondence: [email protected]

Keywords: electrophysiology, modelling, excitotoxicity

Background: Mutant SOD1 (mSOD1) motoneurons in the standard G85R mouse model of ALS exhibit alterations in morphology (1) and electrical properties (2,3), long before symptom onset (P8-P10). These alterations are expected to affect the motoneuronal processing of synaptic inputs and consequently motoneuron recruitment during movement.

Objectives: To assess the individual roles that the changes in morphology, electrical properties, and dendritic ion channels play in the efficacy and summation of synaptic currents in wild-type (WT) and mSOD1 motoneurons.

Methods: We used realistic computer models of WT and mSOD1 lumbar motoneurons in which the motoneuronal electrical and morphological properties could be manipulated independently. Models were based on the reconstructed morphologies of neonatal WT and mSOD1 motoneurons (P8-P10) (1), and model parameters were optimized to match the electrophysiological properties recorded experimentally from the same motoneurons.

Results: Our results from computer simulations indicated that: First, the change in morphological properties does not account fully for the change in electrical properties. Second, the specific membrane resistance (Rm) was reduced in mSOD1 motoneurons by 25% relative to WT. Third, one-third of the reduction in mSOD1 motoneurons input resistance (Rin) relative to WT (2), is due to the morphology change, whereas the other two-thirds are due to the reduction in Rm. Fourth, synaptic efficacy is reduced in mSOD1 motoneurons by 25% (ie −25%), relative to WT. This total change resulted from the concurrent increase in morphology, reduction in Rm, and the increase in dendritic voltage-gated ion channels, which individually accounted for −10% to −15%, −23%, and +25% change in synaptic efficacy, respectively. Finally, the summation of synaptic currents in mSOD1 motoneurons did not exhibit significant differences from that in WT motoneurons despite the alteration in morphology, electrical properties, and synaptic efficacy.

Discussion and conclusions: Our results provide a quantitative analysis of the early alterations in morphology and electrical properties of mSOD1 motoneurons and illustrate the functional consequences of those alterations on the dendritic processing of synaptic inputs. The early reductions in Rin and synaptic efficacy of mSOD1 motoneurons would hinder their recruitment, and could contribute to the later retraction of axons from the neuromuscular junction. This prediction is consistent with the beneficial effects of increased neuromuscular activity that improved motoneuron survival in ALS (4). Our simulations also predict changes in the biophysical properties of mSOD1 motoneurons, and set the stage for new experiments to test these predictions.

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P216 MOTOR UNIT NUMBER ESTIMATION USING THE INCREMENTAL METHOD IN NORMAL DOGS

VASQUEZ L, KANAZONO S, VILLAMIL AJ, DHAND U, O'BRIEN D, COATES J

University of Missouri, Columbia, MO, United States

E-mail address for correspondence: [email protected]

Keywords: canine, degenerative myelopathy, MUNE

Background: Canine degenerative myelopathy (DM) is an adult onset fatal neurodegenerative disease. Initial upper motor neuron paraparesis and general proprioceptive ataxia progress to lower motor neuron tetraparesis. Recently a missense mutation in the canine superoxide dismutase 1 (SOD1) gene has been shown to be a risk factor for DM suggesting homology to human familial amyotrophic lateral sclerosis (ALS) (1). Motor unit number estimation (MUNE) is a technique for quantifying motor unit function and assessing disease progression in ALS patients. If the technique can reliably be established in normal dogs, MUNE could be used as an outcome measure for monitoring therapies in DM affected dogs.

Objective: To adapt the incremental method for MUNE in normal healthy dogs and establish a reference range.

Methods: The incremental stimulation technique described in humans was modified and used on the extensor digitorum brevis (EDB) muscles of 17 normal dogs (age: 1–10 years; body weight 4.5–70 kg) (2). Monopolar stimulating needle electrodes were inserted caudal to the long digital extensor muscle tendon at the level of the tuber calcanei to stimulate the deep peroneal nerve. Direct evoked muscle potentials were recorded with the active surface electrode placed over the EDB motor point and the reference surface electrode over digit IV. The ground electrode was placed subcutaneously between cathode and recording electrode. Supramaximal compound muscle action potential (CMAP) negative peak area was first recorded. Then starting at the subthreshold level, stimulus intensity was slowly increased until the first all-or-none single motor unit potential (SMUP) was evoked. Successive small increments (0.026 mA, 50 μsec) were applied to elicit a total of 10 SMUP responses. The mean SMUP negative peak area was divided into the maximum CMAP negative peak area to yield the MUNE value. Multiple trials were performed on both hindlimbs to assess reproducibility.

Results: The median CMAP area was 4.95 mV·mS (range 1.89–9.32) with 25 and 75 percentile of 3.72 and 5.98, respectively. The median SMUP area was 0.10 mV·mS (range 0.03–0.81) with a 25 and 75 percentile of 0.07 and 0.16, respectively. The MUNE median was 49 (range 8–154) with 25 and 75 percentile values of 30 and 68, respectively. There was no significant difference in MUNE between age groups older and younger than 7 years (P=0.17) or between right and left limbs (P=0.14).

Discussions and conclusions: We demonstrated that EDB incremental method MUNE can be reliably recorded from normal dogs. These results provide potential to apply the described technique for longitudinal monitoring of lower motor neuron signs in DM affected dogs.

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P217 CEREBROSPINAL FLUID NEUROTRANSMITTER CONCENTRATIONS IN CANINE DEGENERATIVE MYELOPATHY; FURTHER SUPPORT OF A CANINE MODEL OF UMN ONSET ALS

PLATT S¹, BARBER R¹, KANAZONO S², EDWARDS G¹, COATES J²

¹College of Veterinary Medicine, University of Georgia, Athens, GA, United States, ²College of Veterinary Medicine, University of Missouri, Columbia, MO, United States

E-mail address for correspondence: [email protected]

Keywords: canine, degenerative myelopathy, neurotransmitters

Background: Recently a missense mutation in the canine superoxide dismutase 1 (SOD1) gene has been shown to be a risk factor for DM suggesting homology to human familial amyotrophic lateral sclerosis (ALS) of UMN onset. Investigations of cerebrospinal fluid (CSF) neurotransmitter concentrations as part of the pathogenesis in human ALS patients suggest a role for excitotoxicity. Treatment strategies for excitotoxicity could be investigated in DM affected dogs if a similar pathogenic role can be suggested.

Objectives: To determine presence or absence of abnormalities in the concentrations of CSF amino acid neurotransmitters: glutamate, glycine and g-aminobutyric acid (GABA) in dogs with DM.

Methods: Forty-six dogs histopathologically confirmed for DM at different clinical stages and 41 clinically normal age-matched control dogs were included in the study. Cerebrospinal fluid was acquired from the cerebellomedullary cistern of dogs while under general anesthesia for diagnostic testing or immediately after euthanasia. All CSF samples were stored at −80°C until time of analysis; 100 μl was deproteinized with methanol (100 μl) and centrifuged at 14,000 g for 2 min. Deproteinized samples were then analysed for presence of glutamate, glycine and GABA by high performance liquid chromatography with fluorescence detection on an Agilent 1100 system following pre-column derivatization with o-phthaldialdehyde (OPA) and 3-mercaptopropionic acid (3-MPA). All analyses were performed using SAS V 9.2 (Cary, NC). Analyte levels were compared between DM affected and age matched control dogs by an analysis of variance (ANOVA). All hypothesis tests were 2-sided with α=0.05.

Results: Glutamate levels were not significantly different between DM affected (mean=0.05 μg/ml; 0.0–0.29; SD=0.069) and control dogs (mean=0.04 μg/ml; 0.0–0.29; SD=0.077). Control dogs (mean=0.94 μg/ml; 0.78–1.16; SD=0.164) had significantly higher levels of GABA (P<0.0001;) than affected dogs (mean=0.10 μg/ml; 0.08–0.19; SD=0.03). Control dogs (mean=2.4 μg/ml; 1.24–5.18; SD=0.82) also had significantly higher glycine concentrations (P<0.0001) than affected dogs (mean=0.4 μg/ml; 0.01–0.78; SD=0.23).

Discussion and conclusions: We demonstrate that DM affected dogs have an imbalance of CSF amino acid concentrations creating a relative excitotoxic environment. Reports in human ALS describe that CSF glutamate is decreased with mild clinical signs, whereas in those with severe clinical signs glutamate will vary from decreased to increased. Similar divergent results have been reported for CSF GABA, glycine and aspartate. An explanation for such differences has been suggested to be to due to disease heterogeneity. Despite these contradictory reports it is believed that an imbalance between CSF excitatory and inhibitory amino acids plays a pathogenic role in ALS. Further prospective analysis of the canine model is warranted to investigate the amino acid variations and possible role as a biomarker at early and late disease stages.

P218 CEREBROSPINAL FLUID FROM PATIENTS WITH SPORADIC ALS – A POTENTIAL TOOL TO DELINEATE THE ETIOPATHOGENESIS OF THE SPORADIC FORM OF THE DISEASE

RAJU TR

National Institute of Mental Health and Neuro Sciences, Bangalore, Karnataka, India

E-mail address for correspondence: [email protected]

Keywords: cerebrospinal fluid, animal model, motor behaviour

Background: The progress in understanding the pathogenesis of sporadic ALS (SALS) has been challenged to a great extent by the absence of appropriate animal models for the disease. Unlike familial ALS, wherein precise genetic mutations have been defined, the SALS form has not been associated to any specific factors.

Objectives: In an attempt to study the etiopathogenesis of SALS, the effect of CSF from SALS patients (ALS-CSF) was tested on motor neurons of the cortex and spinal cord.

Methods: Initially, primary rat embryonic spinal cord cultures were exposed to ALS-CSF and the cellular changes were assessed. Also, to exclude the confounding effects of glia in inducing the changes, we exposed NSC-34 motor neuron cell line to ALS-CSF. To simulate the changes in a more complex system, we intrathecally injected ALS-CSF into neonatal rat pups. Further to determine whether ALS-CSF could induce explicit alterations in motor behaviour and spiking properties of motor neurons, we intracerebroventricularly infused ALS-CSF in adult rats. Local field potentials were recorded from layer V motor cortex in an unrestrained rat. The deficits in motor behaviour were quantified using rotarod and grip strength testing.

Results: ALS-CSF induced degenerative changes in the motor neurons and also affected the astrocytes. Reduced number of Choline acetyl transferase positive spinal motor neurons with aberrant phosphorylation of neurofilaments and enhanced LDH activity were amongst the prominent observations. Ultrastructurally Golgi apparatus were fragmented and numerous small, discrete and unconnected stacks were distributed widely throughout the cytoplasm. Similar changes were reproduced in NSC-34 cells in addition to ubiqitinated protein aggregates and recruitment of endopalsmic reticulum stress responses. Reduced expression of trophic factors indicated an altered microenvironment of motor neurons. Interestingly the expression of sodium and potassium channels on spinal motor neurons was significantly diminished. In line with this, the local field potentials recorded from motor cortex of adult rats showed a bimodal effect on the relative power values. Apart from motor neurons the astrocytes were also affected. Reactive astrogliosis was accompanied by the transformation of astrocytes from flat to fibrous form. Also, the expression of glial glutamate transporter-1 was reduced. These cellular and molecular changes were substantiated by an overt motor behavioral deficit on rotarod and grip strength.

Discussion and conclusion: This is the first comprehensive study to report the effect of ALS-CSF on motor neurons at the molecular, cellular, physiological and behavioral levels. Most of our findings reported here are comparable to those seen in autopsy samples of SALS patients and also animal models of FALS. The degeneration of motor neurons is resultant of the dysfunction of several related mechanisms.