Makbule Senel

MD Tobias A Rupprecht

Hayrettin Tumani
email: hayrettin.tumani@uni-ulm.de

MD Hans W Pfister

MD Albert C Ludolph

MD Johannes Brettschneider

The chemokine CXCL13 in acute neuroborreliosis

Keywords:

Objective: Recent studies have suggested an important role of the B-cell chemoattractant CXCL13 in acute neuroborreliosis (NB). Our aim was to confirm the diagnostic role of CXCL13 and to evaluate its relevance as a therapy response and disease activity marker in NB. Methods: CXCL13 was measured in cerebrospinal fluid (CSF) and serum of patients with NB (NB, n= 28), systemic borreliosis (SB, n=9), Guillain-Barré syndrome (GBS, n=11), Bell's palsy (BP, n=19), other cranial nerve palsies (CNP, n=5), cephalgia (C, n=20), bacterial-CNS-infections (B-CNS-I, n=16) and from patients with viral-CNS-infections (V-CNS-I, n=18). For follow-up studies serial sample pairs were evaluated from 25 patients with NB (n=56), 11 with B-CNS-I (n=25) and 14 with V-CNS-I (n=36). Results: CSF-CXCL13 was significantly elevated in NB compared to other neurological diseases (p<0.001). Using ROC analysis, 337 ng/g was determined as cut-off with a sensitivity of 96.4 % and a specificity of 96.9 %. Of all the parameters investigated, CSF CXCL13 showed the fastest response to antibiotic therapy, decreasing significantly (p=0.008) within one week. In untreated patients, CSF CXCL13 was elevated in patients with a short duration of disease. Borrelia burgdorferi antibody index (BB-AI) showed no significant (p=0.356) change over follow-up. Conclusions: Our study confirms the relevance of CXCL13 as a diagnostic biomarker of NB. It suggests that CSF CXCL13 in NB is linked to duration of disease and could be a marker of disease activity and response to antibiotic therapy.

INTRODUCTION

Neuroborreliosis (NB) is a nervous system manifestation of Lyme borreliosis, caused by the tickborne spirochete Borrelia burgdorferi (BB). [START_REF] Burgdorfer | Lyme disease-a tick-borne spirochetosis[END_REF][START_REF] Benach | Spirochetes isolated from the blood of two patients with Lyme disease[END_REF][START_REF] Steere | The spirochetal etiology of Lyme disease[END_REF][START_REF] Pachner | The triad of neurologic manifestations of Lyme disease: meningitis, cranial neuritis, and radiculoneuritis[END_REF] Due to a great variety of neurological symptoms such as meningitis, meningoradiculitis, or cranial nerve palsies, [START_REF] Pfister | Lyme borreliosis: basic science and clinical aspects[END_REF][START_REF] Hengge | Lyme borreliosis[END_REF] identification of neuroborreliosis frequently is a diagnostic challenge in clinical neurology. Currently the method of choice for the diagnosis of acute NB is the combined analysis of basic cerebrospinal fluid (CSF) parameters (lymphocytic pleocytosis, intrathecal synthesis of immunoglobulins and/ or blood-CSF barrier dysfunction) and determination of BB specific antibody index (BB-AI ≥1.5, i.e. intrathecal antibody synthesis to BB). [7] However, this approach has important limitations: (1) Elevated basic CSF variables are also found in various other inflammatory diseases of the central nervous system (CNS). [START_REF] Reiber | QUANTIFICATION OF VIRUS-SPECIFIC ANTIBODIES IN CEREBROSPINAL FLUID AND SERUM SENSITIVE AND SPECIFIC DETECTION OF ANTIBODY SYNTHESIS IN BRAIN[END_REF][START_REF] Felgenhauer | The diagnostic significance of antibody specificity indices in multiple sclerosis and herpes virus induced diseases of the nervous system[END_REF] (2) Intrathecal BB antibody synthesis may not be detectable in early stages of disease, especially if disease duration is less than 2-3 weeks. [START_REF] Tumani | Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis[END_REF][START_REF] Kaiser | Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto[END_REF][START_REF] Hansen | Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M[END_REF][START_REF] Wilske | Diagnosis of Lyme neuroborreliosis. Detection of intrathecal antibody formation[END_REF][START_REF] Hammers-Berggren | Borrelia burgdorferi-specific intrathecal antibody production in neuroborreliosis: a follow-up study[END_REF] (3) Elevated BB-AI does not discriminate between active and past infections as it is known to persist for years in spite of successful treatment. [START_REF] Hammers-Berggren | Borrelia burgdorferi-specific intrathecal antibody production in neuroborreliosis: a follow-up study[END_REF] (4) False positive results may occur due to antibody cross reactivities. [START_REF] Tumani | Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis[END_REF][START_REF] Hansen | Oligoclonal Borrelia burgdorferi-specific IgG antibodies in cerebrospinal fluid in Lyme neuroborreliosis[END_REF][START_REF] Stiernstedt | Diagnosis of spirochetal meningitis by enzyme-linked immunosorbent assay and indirect immunofluorescence assay in serum and cerebrospinal fluid[END_REF] (5) Due to the use of different isolates as antigen in various immunosorbent assays for BB antibodies, results may vary considerably among different laboratories.

Recent studies have suggested an important role of CXCL13 in the diagnosis of NB. [START_REF] Rupprecht | The chemokine CXCL13 (BLC): a putative diagnostic marker for neuroborreliosis[END_REF][START_REF] Ljostad | CSF B--lymphocyte chemoattractant (CXCL13) in the early diagnosis of acute Lyme neuroborreliosis[END_REF] CXCL13 belongs to the CXC chemokine family and is a selective chemoattractant for B-lymphocytes and Bhelper T-cells via the chemokine receptor CXCR5. [START_REF] Nelson | Chemokines, chemokine receptors, and allograft rejection[END_REF] Our aim was to confirm the diagnostic value of CXCL13 in NB in comparison to established diagnostic parameters such as BB-AI and to evaluate the relevance of CXCL13 as a marker of disease activity and therapy response in acute NB. [START_REF] Reiber | Cerebrospinal fluid--physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases[END_REF]). [START_REF] Reiber | Cerebrospinal fluid--physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases[END_REF] CXCL13 was measured by quantitative enzyme-linked immunosorbent assay (ELISA) (Quantikine; R&D Systems, Minneapolis, MN) according to the instructions as supplied by the manufacturer.

METHODS

Patients

For validating and assessing assay accuracy spike-and-recovery as well as linearity-of-dilution experiments were performed. To compensate for differences in CSF protein due to blood-CSF barrier dysfunction in acute NB, CSF CXCL13 was related to total CSF protein in accordance with Rupprecht et al., Neurology 2005. [START_REF] Rupprecht | The chemokine CXCL13 (BLC): a putative diagnostic marker for neuroborreliosis[END_REF] The Borrelia afzelii+VIsE IgG ELISA (PKo strain, Genzyme Virotech, Rüsselsheim, Germany) was used for detection of IgG antibodies against BB sensu lato.

Statistical analysis

Statistical analysis was performed using SPSS (version 16.0; SPSS Inc., Chicago, IL, USA). Differences of CXCL13 between groups were analyzed using Kruskal-Wallis test followed by single pairwise comparisons (Mann-Whitney U-test). Wilcoxon signed rank test was applied to compare concentrations at individual time points with baseline (diagnostic LP) values, and Friedman test (repeated measures of ANOVA on ranks) was applied to analyse repeated values for a global analysis of progression. Correlation was analysed using Spearman rank order correlation. To test for normality, the Kolmogorov-Smirnov-test was applied. Sensitivity was calculated as (truepositive/ [true-positive + false-negative]), specificity was calculated as (true-negative / [truenegative + false-positive]). To examine the accuracy of CXCL13 to differentiate NB from controls, we used Receiver operating characteristic (ROC) analysis, calculating the area under the ROC curve (AUROCC). The Youden index was calculated for each cut-off value as corresponding [(sensitivity + specificity)] -1] to find the cut-off values which maximize discriminating accuracy. [START_REF] Youden Wj | Index for rating diagnostic tests[END_REF] P values < 0.05 were considered as statistically significant.

RESULTS

Clinical findings and basic CSF examination results

At time of diagnosis, the mean duration of symptoms in NB patients was 11 days (median 7; range 1 to 40). Only 32.1% of patients (9 of 28) remembered a tick bite. The most frequent neurological symptoms in NB patients were polyradiculitis (9 of 28) and facial nerve palsy (10 of 28). In CSF, all patients with NB showed a lymphocytic pleocytosis and most of the patients (82 %) revealed increased CSF protein measured by an elevation of Q alb (table 1). Intrathecal IgM synthesis was found in 78%, while IgG and IgA synthesis were observed in 35.7 % (table 1). All NB patients showed elevated BB-IgG-AI (≥1.5), with a median AI of 17 (range 2.6-104, table 1).

Assay validation ELISA matrix spike recovery was acceptable, with an average recovery of 100% and a range in spike recoveries of 93-109% (n=9). Linearity-of-dilution experiments showed an average recovery of 102.5 % (range 98-109 n=6). According to linearity-of-dilution experiments concentrations < 3.9 pg/ml were stated as not detectable.

CXCL13 at diagnostic lumbar puncture

Patients with NB showed significantly elevated CSF CXCL13 levels as compared to all other groups (p= 0.001, figure 1). NB patients with a shorter duration of disease (≤ 8 days) showed significantly higher CSF-CXCL13 concentrations as compared to patients with a longer duration of disease (> 8 days) (pair wise comparison,p=0.016; scatterplot, figure 3). Higher CSF CXCL-13 concentrations in those patients tended to be associated with higher CSF leukocyte count (p = 0.095), though this did not reach statistical significance. CSF-CXCL13 correlated with neither Q alb (R=0.350 p=0.068) nor with serum-CXCL13 (R=0.372 p=0.067). CSF CXCL13 levels were slightly elevated in single cases of bacterial or viral CNS infections, with highest values being detected in patients with neurosyphilis (NS), Staphylococcus aureus meningomyelitis and meningoencephalitis caused by HSV (figure 1). CXCL13 was under the detection limit in most patients with cephalgia, BP, CNP, GBS and patients with SB (figure 1, table 1). Serum-CXCL13 was elevated in B-CNS-D (inclusive NS) but was otherwise not significantly different in NB as compared to controls (figure 2, table1). Highest serum-CXCL13 was observed in patients with pneumococcal or Haemophilus influenza infections. The mean CXCL13 CSF/serum ratio in NB was 81.9, which was significantly higher as in all other groups (p< 0.001). Two weeks after onset of antibiotic therapy, 41% of NB patients showed a complete remission of symptoms, 53% reported subjective complaints such as pain or paresthesia, and 6% showed objective deficits such as persisting Bell's palsy. CXCL13 showed a significant decrease within one week (p=0.008) of onset of antibiotic therapy (figure 4), with CSF CXCL13 levels decreasing below the cut-off after two weeks. In contrast, total CSF leukocyte count decreased significantly (p=0.009) only during the second week after onset of antibiotic treatment, though CSF leukocyte count still remained above the normal level (>4/µl) in all patients. BB-AI showed no significant changes over follow-up (p=0.356). Of all investigated parameters, CXCL13 showed the fastest and most pronounced decrease after onset of antibiotic therapy. In detail, CSF-CXCL13 fell considerably in all 25 NB-patients whereas CSF leukocyte count (in 28%), albumin ratio (in 12%) and total protein (in 20%) showed even an increase in some patients.

As control groups, we analyzed serial samples from patients with bacterial (n=11) and viral (n=14) meningitis/meningoencephalitis. CSF CXCL13 levels in bacterial infections of the CNS showed no significant change over follow-up (p=0.112) whereas CSF leukocyte count (in 81% of patients), albumin ratio (in all) and lactate (in all) decreased. The CSF CXCL13 levels in viral infections of the CNS showed also no significant change during follow-up (p=0.448), as did leukocyte count and Q alb . Serum CXCL13 levels were similar to serum CXCL13 levels of controls.

DISCUSSION

CXCL13 as diagnostic marker in neuroborreliosis

We observed elevated CSF-CXCL13 in NB and neurosyphilis (NS) as compared to other CNSdiseases, which is in line with previous studies. [START_REF] Ljostad | CSF B--lymphocyte chemoattractant (CXCL13) in the early diagnosis of acute Lyme neuroborreliosis[END_REF][START_REF] Rupprecht | Cytokine CXCL13--a possible early CSF marker for neuroborreliosis[END_REF][START_REF] Rupprecht | Borrelia garinii induces CXCL13 production in human monocytes through Toll-like receptor 2[END_REF] At a cut-off of 337ng/g, CSF-CXCL13 revealed the highest combined sensitivity (96.4%) and specificity (96.9%) for diagnosis of NB of all parameters investigated. Basic CSF findings had an acceptable sensitivity (96.4%) but insufficient specificity (63.3%). Due to our selection criteria of patients with NB sensitivity of BB-AI was 100%, but other studies reported BB-AI to be negative in about 10-20% at early stages of disease activity (disease duration less than 2-3 weeks). [START_REF] Tumani | Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis[END_REF][START_REF] Kaiser | Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto[END_REF][START_REF] Hansen | Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M[END_REF][START_REF] Wilske | Diagnosis of Lyme neuroborreliosis. Detection of intrathecal antibody formation[END_REF][START_REF] Hammers-Berggren | Borrelia burgdorferi-specific intrathecal antibody production in neuroborreliosis: a follow-up study[END_REF] Furthermore, our data revealed specificity of BB-AI to be comparatively low (87.1%), which may be due to past infections with BB or antibody crossreactivity with other infectious agents such as Treponema pallidum. Combination of BB-AI and basic CSF variables improved diagnostic accuracy in accordance with previous studies [START_REF] Tumani | Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis[END_REF] but did not reach diagnostic accuracy of CXCL13.

Only in three patients with diagnosis other than NB elevated CSF-CXCL13 was observed: One patient was diagnosed as HSV encephalitis, one as neurosyphilis and one suffered from Staphylococcus aureus meningoencephalitis. We observed no correlation of CSF CXCL13 with either Q alb or serum CXCL13, indicating that CSF CXCL13 is mainly synthesized intrathecally. Serum CXCL13 was not useful for diagnosis of NB but showed elevated levels in patients with B-CNS-D. Consequently, serum-CXCL13 could be useful to differentiate between NB and other B-CNS-D, especially in the case of NS, which was also observed to show elevated CSF-CXCL13 concentrations. [START_REF] Rupprecht | Borrelia garinii induces CXCL13 production in human monocytes through Toll-like receptor 2[END_REF] The pathophysiological mechanisms underlying this discrepancy of CSF and serum CXCL13 in NB are a matter of speculation. Immune evasion strategies of BB in the blood compartment like downregulation of outer surface proteins (Osp), hiding in extracellular matrix, or the secretion of complement-neutralizing proteins was suggested to reduce the immune response to BB in the peripheral blood and consequently the production of CXCL13 by monocytes and dendritic cells. [START_REF] Rupprecht | The pathogenesis of lyme neuroborreliosis: from infection to inflammation[END_REF] Furthermore, migration of BB into the CNS along peripheral nerves could also enable BB to avoid the host immune system, resulting in low serum CXCL13 levels. [START_REF] Horstrup | Tick born meningopolyneuritis (Garin-Bujadoux, Bannwarth) (author's transl)[END_REF][START_REF] Rupprecht | The pathogenesis of lyme neuroborreliosis: from infection to inflammation[END_REF] In the nervous system, OspA has been observed to be upregulated again, inducing the secretion of CXCL13 by interaction with the toll-like receptor 2 of monocytes. [START_REF] Rupprecht | The pathogenesis of lyme neuroborreliosis: from infection to inflammation[END_REF] CXCL13 as marker of disease activity and duration Of all the parameters investigated, CSF CXCL13 showed the fastest response to antibiotic therapy with intravenous ceftriaxone, decreasing significantly within one week (figure 4). In contrast, BB-AI showed no significant decrease under therapy, which is in line with previous studies that found BB-AI to persist up to several years after acute NB. [START_REF] Tumani | Relevance of cerebrospinal fluid variables for early diagnosis of neuroborreliosis[END_REF][START_REF] Kaiser | Analysis of the intrathecal immune response in neuroborreliosis to a sonicate antigen and three recombinant antigens of Borrelia burgdorferi sensu stricto[END_REF][START_REF] Hansen | Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M[END_REF][START_REF] Wilske | Diagnosis of Lyme neuroborreliosis. Detection of intrathecal antibody formation[END_REF][START_REF] Hammers-Berggren | Borrelia burgdorferi-specific intrathecal antibody production in neuroborreliosis: a follow-up study[END_REF] Accordingly, BB-AI could mimic acute inflammation with BB, especially in cases of elevated cell count due to an infection with another pathogen. The fast decline under therapy with ceftriaxone suggests that CSF CXCL13 may offer a new tool to measure disease activity in patients with NB. This is in line with a previous study that reported a strong correlation between CXCL13 gene expression and spirochetal load. [START_REF] Narayan | The nervous system as ectopic germinal center: CXCL13 and IgG in lyme neuroborreliosis[END_REF] However, we also observed high levels of CSF CXCL13 to be related to a short duration of disease prior to lumbar puncture, suggesting that CSF CXCL13 may be a marker of disease duration in NB rather than a marker of disease activity. Future studies including a larger number of patients with follow-up data will have to clarify whether CXCL13 is a marker of disease activity or merely a marker of disease duration in NB.

Conclusion

Our study confirms the relevance of CXCL13 as a diagnostic biomarker of NB. It suggests that CSF CXCL13 levels in NB are linked to duration of disease and could be a marker of disease activity and response to antibiotic therapy. We recommend CSF-CXCL13 to be used for diagnosis of NB in addition to established parameters like BB-AI. Especially in cases with positive BB-AI due to past NB and elevated CSF leukocyte count due to an infection with another pathogen, CSF-CXCL13 is currently the best tool to differentiate between active and past infection with BB.

  Demographic data and basic CSF findings of patients included in this study. Data are shown as median and interquartile range; NB = neuroborreliosis, SB= systemic borreliosis, B-CNS-D = bacterial infections of the central nervous system, V-CNS-D = viral infections of the central nervous system, C = cephalgia, GBS = Guillain-Barré syndrome, CNP = cranial nerve palsies, BP = Bell's palsy. Qalb = CSF/serum albumin concentration ratio, IgM synthesis is intrathecal IgM synthesis, OCB = oligoclonal IgG bands.

Figure 1 :

 1 Figure 1: CXCL13 (ng/g protein) in the CSF of neuroborreliosis and other neurological diseases; horizontal solid lines indicate medians. Kruskal-Wallis-test and pair wise comparisons (Mann-Whitney U-test) revealed a significant difference between the groups (p<0•001). Dashed line at 337ng/g represents the cut-off level yielding the highest combined sensitivity and specificity for discriminating patients with NB form those with other diseases. Gray triangle indicates an outlier (neurosyphilis patient) with CSF CXCL13 of 113000 ng/g. yaxis is broken twice; NB = definite neuroborreliosis, SB = systemic borreliosis, B-CNS-D = bacterial CNS disease, V-CNS-D = viral CNS disease, C = cephalgia, GBS = Guillain-Barré-Syndrom, CNP = cranial nerve palsies, BP = Bell's palsy.

Figure 2 :

 2 Figure 2: Serum CXCL13 (pg/ml) in bacterial-CNS-disease (B-CNS-D), neuroborreliosis (NB) and other neurological diseases; Kruskal-Wallis-test p< 0.001, pair wise comparisons (Mann-Whitney U-test) p<0.01; for explanation of abbreviations see Figure 1.

Figure 3 :

 3 Figure 3: CSF CXCL13 in neuroborreliosis patients prior to therapy plotted against time since onset of symptoms (time to lumbar puncture). Straight line is regression line; correlation was significant (p = 0.044, R = -0.383).

Figure 4 :

 4 Figure 4: Relative changes (median and IQR) in CSF CXCL13, leukocyte count, Q(albumin) (CSF/serum albumin concentration ratio) and BB-IgG-AI over follow-up in 25 patients with neuroborreliosis.

Table

  Serum sample pairs from 25 patients with NB (n=56), 11 patients with bacterial (n=25) and 14 patients with viral (n=36) infections of the CNS. Samples of eight of these NB patients were obtained from the Department of Neurology, Ludwig-Maximilians University, Munich, Germany. Standard antibiotic therapy of NB patients consisted of ceftriaxone 2 g intravenously per day over 2 weeks.CSF analysis and CXCL13 assayAll CSF samples were stored at -80° C until analysis. CSF leukocyte count (cells/cu.mm), total protein (g/L), lactate (mmol/L), CSF/serum albumin concentration ratio (Q alb ), immunoglobulin (Ig) G, A and M and oligoclonal IgG bands (OCB) were obtained as previously described.[START_REF] Sussmuth | Amyotrophic lateral sclerosis: disease stage related changes of tau protein and S100 beta in cerebrospinal fluid and creatine kinase in serum[END_REF] Intrathecal synthesis of antibodies to BB was determined by calculation of the specific antibody indices (AI): AI = QIg[spec]/QIg[total], if QIg[total] < Qlim, and AI = QIg[spec]/Qlim, if QIg[total] > Qlim.[START_REF] Reiber | Cerebrospinal fluid--physiology, analysis and interpretation of protein patterns for diagnosis of neurological diseases[END_REF] The upper reference range of QIg[total], Qlim, was calculated according to Reiber's formula. AI values ≥ 1.5 were considered to be indicative of intrathecal Ig synthesis against BB

	Drawing of blood and lumbar puncture was performed for diagnostic purposes after the patient's
	informed consent was obtained. Paired CSF and serum samples were collected between January
	2000 and September 2008 from 126 patients admitted to the Department of Neurology, University
	of Ulm, Germany (for demographic data, see table 1). Patients included 28 patients with definite
	neuroborreliosis (NB) according to criteria by Kaiser[7] and 9 patients with systemic borreliosis
	(SB) who showed no clinical signs of neuroborreliosis and showed normal basic CSF findings
	(including cell count, total protein and lactate). At time of lumbar puncture, none of the NB
	n (f/m) patients received any antibiotic therapy. Furthermore, we included 69 patients with neurological Age [years] Cell count [/µl] Qalb (x 10 -3 ) IgM OCB [%] diseases relevant in the differential diagnosis of NB, comprising of 16 patients with bacterial synthesis [%] infections of the CNS (B-CNS-D, including meningitis or meningoencephalitis caused by Treponema
	pallidum, Mycobacterium tuberculosis, Streptococcus pneumoniae, Meningococcus, Listeria
	NB monocytogenes, Haemophilus influenzae, Staphylococcus aureus or other Streptococci), 18 28 (9/19) 58 (32-70) 84 (50-187) 13.8 (9.7-23.3) 78.6	85.7
	SB patients with viral CNS diseases (V-CNS-D, including meningitis or meningoencephalitis caused by 9 (1/8) 44 (40-56) 1 (1-4) 6•6 (4.6-8.3) 22•2 11.1
	B-CNS-D herpes simplex virus (HSV), varicella zoster virus (VZV), Epstein-Barr virus (EBV), rubella virus, tick-16 (9/7) 53 (36-69) 2134 (117-4682) 58.1 (35.5-90.2) 18.8 13.3 borne encephalitis Virus (TBEV), enterovirus), 11 patients with Guillain-Barré syndrome (GBS), 19
	V-CNS-D 18 (3/15) 38 (27-63) 178 (54-290) patients with Bell's palsy (BP) and 5 patients with other cranial nerve palsies (CNP). As normal 11.8 (8.8-16.7) 16.7 22.2
	C controls, we included 20 patients with cephalgia (C) who presented with tension-type headache 20 (10/10) 53 (44-64) 1 (0-2) 4.8 (3.7-7.1) 0 0
	and showed no evidence of a structural, hemorrhagic or inflammatory lesion.
	GBS For follow-up studies we included serial CSF/11 (4/7) 40 (27-51) 1 (0-2)	17.4 (6.8-23.2) 9.1	63.6
	CNP	5 (1/4)	65 (54-66) 2 (1-2)	6.2 (5.7-8.4)	0	0
	BP	19 (11/8) 40 (26-67) 2 (1-3)	5.2 (4.4-6.0)	0	15.8

Table 2 :

 2 Accuracy of CSF markers for diagnosis of neuroborreliosis; sensitivity and specificity in percent (95% confidence interval in brackets); basic CSF findings (BCF) = pleocytosis > 4 cells, intrathecal IgM synthesis and/or elevation of Q alb ; BB-AI is AI ≥ 1•5.

	Sensitivity	Specificity
	Basic CSF findings	96.4 (82-100)	63.3 (53-73)

showed a sensitivity of 96.4% and a specificity of 63.3% (table

2

). Combination of pathological BB-AI and basic CSF variables yielded a sensitivity of 96.4% and a specificity of 90.3% (table

2

). CSF-CXCL13 was elevated (>337ng/g) in only three cases with inflammatory CNS reaction not due to BB, including a patient with neurosyphilis, Staphylococcus aureus meningomyelitis and HSV meningoencephalitis. Only in one patient with NB CSF CXCL13 was below 337 ng/g. BB-IgG-AI was positive (≥ 1.5) in four patients with diagnoses other than NB.

CXCL13 over follow-up

Acknowledgements

We thank Dr Vera Lehmensiek and Mrs. Helga Mogel for their technical assistance. The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in PMJ and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence. Competing interest: None declared.


Daniel Weiss

Tobias Wächter

Sorin Breit

Simon N Jacob

Jörn K Pomper

Friedrich Asmus

Josep Valls-Solé

Christian Plewnia

Thomas Gasser

Alireza Gharabaghi

Rejko Krüger
email: rejko.krueger@uni-tuebingen.de

Involuntary eyelid closure after STN-DBS: evidence for different pathophysiological entities

Keywords: deep brain stimulation (DBS), subthalamic nucleus (STN), blepharospasm, apraxia of lid opening (ALO), Parkinson´s disease (PD)

Objective: Involuntary eyelid closure (IEC) may occur after deep brain stimulation of the subthalamic nucleus (STN-DBS) in Parkinson´s disease (PD) and is often categorised as apraxia of lid opening (ALO), albeit the appropriateness of this term is under debate. To gain insight into the hitherto undefined pathophysiology of IEC after STN-DBS we performed a comprehensive clinical and electrophysiological characterisation of lid function in a total of six PD patients.

Methods:

The study was carried out in six PD patients who developed IEC after STN-DBS.

They underwent neurological examination and electromyography recording of activity in the orbicularis oculi muscle (OO) upon varying stimulation patterns. Intraoperative studies were performed in one patient.

Results: Increasing STN-DBS intensity induced IEC in four patients, whereas it improved the condition in two. Needle EMG showed tonic hyperactivity of the OO in STN-DBS induced IEC, while variable patterns of OO activity (irregular and tonic) were seen in patients with STN-DBS relieved IEC. Intraoperative analysis in one patient showed evidence for IEC being induced by activation of corticobulbar fibres.

Conclusions:

We identified two groups of IEC after STN-DBS based on clinical and EMG patterns: (i) STN-DBS induced IEC associated with tonic OO overactivity and (ii) STN-DBS relieved IEC presenting with variable EMG patterns. Our findings provide relevant information on pathophysiology of STN-DBS related IEC and implications for its therapeutic management.

Introduction

Therapeutic deep brain stimulation of the subthalamic nucleus (STN-DBS) provides an effective evidence-based symptomatic treatment for advanced Parkinson´s disease (PD) [START_REF] Kleiner-Fisman | Subthalamic nucleus deep brain stimulation: summary and meta-analysis of outcomes[END_REF][START_REF] Krack | Five-year follow-up of bilateral stimulation of the subthalamic nucleus in advanced Parkinson's disease[END_REF][START_REF] Rodriguez-Oroz | Bilateral deep brain stimulation in Parkinson's disease: a multicentre study with 4 years follow-up[END_REF] . Optimal hardware programming can be challenging in some cases, as side effects may interfere with stimulation parameters required for sufficient control of motor symptoms [START_REF] Deuschl | Deep brain stimulation: postoperative issues[END_REF] . A common and limiting side effect is involuntary eyelid closure (IEC) sometimes referred to as apraxia of lid opening (ALO). This specific symptom has been reported in 1.8 to 30 % followed up after STN-DBS [START_REF] Kleiner-Fisman | Subthalamic nucleus deep brain stimulation: summary and meta-analysis of outcomes[END_REF][START_REF] Deuschl | Deep brain stimulation: postoperative issues[END_REF][START_REF] Hariz | Multicenter study on deep brain stimulation in Parkinson's disease: an independent assessment of reported adverse events at 4 years[END_REF] .

Goldstein and Cogan defined ALO in 1965 as the patient´s inability to voluntarily initiate opening of eyelids [START_REF] Goldstein | Apraxia of Lid Opening[END_REF] . ALO was specified clinically as an IEC without ongoing visible contraction of the orbicularis oculi muscle (OO) and without oculomotor nerve dysfunction or ocular, myopathic or myasthenic signs. Enhanced activation of the frontalis muscle during periods of inability to open the eyes voluntarily has been observed presumably indicating insufficient compensatory attempts of activating neighbouring cranial muscles [START_REF] Goldstein | Apraxia of Lid Opening[END_REF][START_REF] Lepore | Apraxia" of eyelid opening: an involuntary levator inhibition[END_REF] . However, the terminology of ALO is under debate, as some authors do not consider it as "true apraxia" but rather as a form of focal dystonia [START_REF] Krack | Apraxia of lid opening," a focal eyelid dystonia: clinical study of 32 patients[END_REF] . Recent systematic EMG studies show failure of voluntary activation of the levator palpebrae superioris (LPS) during periods of eye closure [START_REF] Reichel | Palpebral Blepharospasm -Distinction from Apraxia of Lid Opening and Disturbance of Inhibition by Synchronous Electromygraphy[END_REF][START_REF] Esteban | Involuntary closure of eyelids in parkinsonism. Electrophysiological evidence for prolonged inhibition of the levator palpebrae muscles[END_REF][START_REF] Aramideh | Electromyographic features of levator palpebrae superioris and orbicularis oculi muscles in blepharospasm[END_REF][START_REF] Esteban | Eyelid movements in health and disease. The supranuclear impairment of the palpebral motility[END_REF] . Replacement of ALO by the terms "inhibition of levator palpebrae superioris muscle" or "blepharocolysis" has been suggested accordingly. Tonic activity of the OO and inhibition of LPS even co-occurred within a single patient due to impaired antagonistic inhibition [START_REF] Reichel | Palpebral Blepharospasm -Distinction from Apraxia of Lid Opening and Disturbance of Inhibition by Synchronous Electromygraphy[END_REF][START_REF] Aramideh | Electromyographic features of levator palpebrae superioris and orbicularis oculi muscles in blepharospasm[END_REF] .

Here, we present a case series of six PD patients exhibiting clinical features of IEC in the postoperative management of STN-DBS. The rationale for our study was to define electrophysiological signatures in these patients and to investigate whether IEC after STN-DBS matched the clinical criteria of previously described ALO or blepharospasm upon clinical and electrophysiological characterization. Additionally, testing of different stimulation parameters in correlation with surgery protocols including intraoperative test stimulation and microelectrode recordings, as well as testing of response to levodopa, allowed us to define pathophysiological and topographical characteristics of IEC. These findings were supported by one additional case with intraoperative analysis of stimulation induced IEC. Our results enable to categorise IEC in PD patients after STN-DBS into different groups with direct implications for its therapeutic management.

Patients and methods

General information on patients

We reviewed 83 consecutive records of PD patients treated with STN-DBS in Tübingen. IEC was reported in 9.6 % (n = 8). Six of these eight PD patients (PD1 -PD6; six male; mean age 65.7 ± 6.0 years; mean duration of disease 17.9 ± 4.9; preoperative Hoehn & Yahr stage ranging from 4 to 5) were available for further clinical evaluation (clinical details in table 1). In one patient (PD4), only data from stimulation through the right hemisphere electrodes were used for the analysis because of intraoperative bleeding at the left hemisphere electrode location. All six patients (PD1-PD6) experienced IEC after bilateral implant of STN-DBS electrodes leading to intermittent functional blindness. They were scheduled for a systematic clinical examination and specific electrophysiological tests (see below).

In addition, we recorded intraoperative data in one patient (PD7), who displayed IEC during intraoperative test stimulation of the right STN area allowing a comprehensive topographic mapping of potentially involved neural structures.

Surgical procedure

Patients underwent bilateral electrode implantation into the STN using the CRW stereotactic frame (Radionics, Burlington, MA) between 2003 and 2009. The target point was located 12 mm lateral, 3-4 mm posterior and 4-5 mm inferior to MCP. The trajectories had an double-oblique orientation entering the STN from anterio-lateral. Postoperative imaging confirmed the location of the active contacts within the STN. The orientation of the contact that modulated IEC at lowest stimulation intensity was determined in comparison to the target point (supplemental table 1). All patients were operated with local anaesthesia, only one patient (PD2) was subjected to general anaesthesia at the beginning of the surgical procedure due to severe clinical symptoms of levodopa discontinuation and thus was tested for direct side effects of test stimulation.

Electrophysiological exploration of the targeted brain area was performed using three microrecording / macrostimulation electrodes (FHC, Bowdoinham, ME). Clinical testing along trajectories for therapeutic and side effects was performed afterwards. The quadripolar electrode (type 3389, Medtronic, Minneapolis, MN) was inserted into each STN along the trajectory that yielded best results in electrophysiological exploration and test stimulation and connected to an implantable pulse generator (Kinetra, Medtronic, Minneapolis, MN) (supplemental table 1). All patients received post-operative MRI (T1, 1.5 Tesla-Scanner, Siemens Sonata, Erlangen, Germany) according to the manufacturers guidelines (Medtronic, USA; SAR < 0.1 W/kg) to ensure correct electrode placement one day after bilateral implantation of chronic electrodes.

Clinical and electrophysiological features

We used clinical and electrophysiological tests to characterise IEC in our patients. We specifically assessed (i) the effects of STN-DBS on IEC (induction vs. reversal) and (ii) the pattern of OO-EMG activity associated with IEC.

Clinical testing for the effects of changing STN-DBS parameters on IEC Stimulation parameters were tested systematically. Every cathodal monopolar contact was tested with standard parameters (pulse width: 60 µs; frequency: 130 Hz) with contralateral STN contacts set to 0 V. Voltage was subsequently increased in intervals of 0.5 V and beneficial effects as well as stimulation-induced side effects were recorded by a neurologist specialised in DBS (DW). The thresholds for elicitation of IEC for all electrode contacts in each patient were determined to specify the spatial representation of this side effect. Electrodes were labelled according to the consensus nomenclature for Kinetra ® stimulators (0-3: left STN; 4-7: right STN). 0 and 4 indicated the most distal, 3 and 7 the most proximal contacts of electrodes on the left and right side, respectively.

Electromyographic testing of the orbicularis oculi muscle (OO)

In patients PD1 to PD6, we performed needle electromyography testing of the orbicular part of the right lower OO using concentric electromyography needles (25 mm length, 0.3 mm diameter, Ambu Neuroline, Ballerup, Denmark). The localisation of the needle was controlled by voluntary forceful eyelid closure resulting in tonic OO discharge in all of the patients. The OO-EMG was recorded with stimulators on and off with straight forward gaze resulting in two recording conditions in each patient: A) StraightOFF, B) StraightON. EMG recordings were performed with NeuroScreen plus (Jaeger-Toennies, Würzburg, Germany) and sampled at 9615 Hz. To allow data evaluation in the stimulator 'on' condition, all data were exported to Matlab 7.0 and band-pass filtered from 10-120 Hz resulting in adequate reduction of stimulation artefacts.

Innervation patterns were determined after filtering.

Data analysis

Filtered OO-EMG data were inspected and categorised among discharge patterns in direct comparison of inner subject data with and without IEC (background activity, tonic overactivity, irregular overactivity). Subsequently, amplitudes of the OO-EMG activity were quantified by root mean square analysis according to equation 1.

(eq. 1)

Results

Clinical testing for the effects of stimulation

Clinical testing could differentiate two groups: (i) group 1 (n=4; PD1-PD4) in which bilateral IEC could be induced by unilateral STN-DBS, and (ii) group 2 (n=2; PD5, PD6) in which IEC was reversed by STN-DBS. Individual parameters of all patients when IEC occurred for the first time and the parameters that improved IEC are listed in detail below (supplemental table 2).

IEC induced by stimulation

The first documentation of IEC induced by STN-DBS in patients PD1 to PD4 ranged from a few days to one year after STN-DBS surgery. As indicated in the methods section, a total of seven electrodes were available for testing in these four patients. Unilateral stimulation on monopolar contacts was capable to induce bilateral IEC in all of these patients. Threshold amplitudes varied from 1.0 -6.5 V on standard parameters among patients, electrode contact and STN-sides (table 2). In five out of seven electrodes tested, IEC was elicited with lower thresholds on the more distal contacts with a mean gradient of -2.3 V (SD = 1.3 V). Stimulation thresholds for typical side effects due to activation of capsular fibres (i.e. visible muscular contraction) were evoked at comparable intensity as IEC (with a mean difference of less than 1.0 V) and showed similar rostro-caudal distribution of threshold amplitudes (table 2). IEC was preceeded in two patients by increased rates of involuntary blinks (PD1 and PD3), and in one patient by prolonged involuntary blinks (PD2) at stimulation intensities that were subthreshold for onset of permanent IEC. In PD4, no such premonitory clinical signs were observed.

IEC improved by stimulation

Patients PD5-PD6 showed a reversal of IEC when increasing STN-DBS intensity.

In patient PD5, IEC first appeared after reduction of dopaminergic medication post surgery (44% reduction of levodopa equivalent dose compared to preoperative baseline) and worsened over time in line with a decrease in the intensity of STN-DBS. In episodes with IEC eyelids could be opened passively by the physician and only closed with onset of the next involuntary or voluntary eye blink. The patient described that IEC was milder, if environment or situation required attention. In contrast, gazing into light provoked IEC. Clinically, compensatory contractions of the frontalis muscle were observed when the patient tried to open his eyelids voluntarily in episodes of IEC. IEC was completely reversed within seconds after onset of unilateral STN-DBS and reappeared within a minute after switching off the stimulus. Contacts (1, 5, 6) were the most effective for improvement of IEC (table 2). IEC in PD6 first occurred when the STN-DBS accidentally switched off three years after surgery.

IEC appeared within a few minutes and resulted in visible tonic contractions of the OO and in great difficulties for passively moving the eyelids. IEC completely resolved within seconds after switching on the stimulator with contacts 2 and 6 being most effective (table 2).

The most beneficial contacts for improvement of IEC in all of the two patients had also proven In all patients with IEC improved by stimulation (PD5, PD6), additionally the influence of oral application of levodopa (200 mg) on IEC was tested. Therefore, all dopaminergic medication was withdrawn over night (at least 12 hours) and STN-DBS was turned off at least 30 minutes before.

Medical testing was performed after 30 -60 minutes of application and clinical symptoms were rated using the UPDRS III. In PD5, IEC completely reversed 40 minutes after the single dose and dopaminergic on-state was documented by UPDRS III scores (MedOff/StimOff: 42; MedOn/StimOff: 27). In PD6, levodopa reversed IEC completely within 30 minutes after intake (UPDRS III MedOff/StimOff: 57; MedOn/StimOff 25). IEC reappeared 2.5 -3 h after oral application of levodopa related to subsequent wearing-off.

Electromyographic recording from the OO

EMG recordings defined different electrophysiological groups.

In four of the six patients (PD1-PD4) background activity of the OO was present during stimulator off. A tonic interference pattern of the OO occurred in presence of IEC during stimulation (see supplemental figure 1). Consistently, root mean square analysis indicated an increase of activity upon stimulation (table 3).

In PD5, irregular discharge of the OO was observed during IEC in the stimulator off condition (see supplemental figure 2). Activity significantly decreased between single bursts of the OO.

Despite of the transient reduction of OO-EMG discharge, the patient was not able to open eyelids voluntarily. This irregular pattern remained unchanged if the patient was advised to forcefully try

to open his eyes during IEC. Stimulation completely reversed the irregular overactivity of the OO and allowed voluntary eyelid opening. In correlation, the root mean square analysis demonstrated a decrease of OO activity during stimulation (table 3).

In PD6, OO-EMG revealed a tonic interference pattern with increased amplitudes during IEC in the stimulator off condition. Subsequent stimulation reversed IEC completely (see supplemental figure 3) and OO-EMG amplitudes decreased (table 3). OO-EMG pattern in this patient was inverse to the pattern observed in patients PD1 to PD4.

IEC during intraoperative test stimulation

In PD7, bilateral IEC occurred during intraoperative test stimulation of the right STN and could be induced at 2.0 mA (pulse width: 60 µs, frequency: 130 Hz) on the central trajectory on the anatomic target point. Test stimulation on the medial trajectory induced IEC at 4.0 mA (see video). Further clinical signs for stimulation of capsular fibres included dysarthria at 1.5 mA (central trajectory) and at 3.5 mA (medial trajectory) (detailed information on intraoperative effects from test stimulation are available in supplemental table 3).

Discussion

The present study for the first time correlates clinical observations with electromyographic patterns in patients with IEC after STN-DBS. Our results indicate that IEC in PD patients after STN-DBS might have a complex pathophysiology and that the previously applied term ALO, referred to non-paralytic motor abnormality, falls short of the clinical heterogeneity in these patients. Our results underline the relevance of proper clinical and electrophysiological categorisation to initiate adequate therapeutic management.

Detailed analyses of clinical data allowed the definition of two groups: (i) group 1 with IEC induced by stimulation, and (ii) group 2 with IEC improved by stimulation or levodopa. All subjects of group 1 showed a tonic interference EMG pattern and increase of OO-EMG activity during IEC as commonly observed in blepharospasm, either idiopathic or secondary [START_REF] Aramideh | Electromyographic features of levator palpebrae superioris and orbicularis oculi muscles in blepharospasm[END_REF] . In contrast, patients of group 2 presented with variable electrophysiological features: PD5 displayed irregular overactivity of the OO. Instead, PD6 showed an inverse pattern with respect to patients of group 1, with tonic OO discharge during IEC that was clinically and electrophysiologically reversed by stimulation or levodopa.

IEC induced by STN-DBS showed a characteristic distribution of threshold intensities with caudal contacts eliciting IEC at lower stimulation intensities than rostral ones. Caudal electrode contacts are located in general more medial but also closer to the capsula interna that descends and approaches laterally to the STN. Therefore, we speculate on corticobulbar tracts as the neuroanatomical substrates of the observed activation of the OO muscle. This is in line with previous studies showing that STN-DBS can activate cortico-bulbar and other neighbouring tracts: single pulse STN-DBS caused facilitation of the R1-and inhibition of the R2-components of the blink reflex to trigeminal nerve stimulation due to prepulse conditioning, presumably because of activation of fibres in the pallido-pedunculopontine tract [START_REF] Costa | Single subthalamic nucleus deep brain stimuli inhibit the blink reflex in Parkinson's disease patients[END_REF] . Unilateral single-pulse STN-DBS and transcranial magnetic stimulation (TMS) induced bilateral activation of cranial nerve innervated muscles including OO, suggesting that fibres to both ipsi-and contralateral motor nuclei descended together at the level of the STN [START_REF] Costa | Motor responses of muscles supplied by cranial nerves to subthalamic nucleus deep brain stimuli[END_REF][START_REF] Benecke | Transcranial magnetic stimulation of the human brain: responses in muscles supplied by cranial nerves[END_REF] .. In our patients, unilateral stimulation caused bilateral IEC due to tonic overactivity of the OO. Therefore, the dorsal intermedial facial nucleus innervating the OO could be the structure mediating our findings, as it receives bilateral projections from the capsula interna. Unintended activation of descending cortico-bulbar fibre tracts might cause either direct depolarisation of the nuclei or facilitation of excitatory postsynaptic potentials generated in the motoneurons from other concomitant inputs.

Especially, the lower stimulation threshold for bilateral IEC on the more lateral trajectory in PD7 and similar thresholds for side effects mediated through stimulation of the capsula interna in PD1-PD4 would be compatible with this hypothesis. A further intraoperative case report by Shields and colleagues also reported bilateral IEC from stimulation but speculated on a premotor inhibition of the central caudal nucleus (innervating LPS) mediated by trigeminothalamic fibre tracts [START_REF] Shields | Eyelid apraxia associated with subthalamic nucleus deep brain stimulation[END_REF] . However, tonic overactivity of the OO in our study would not comply with this explanation. Current spreading to structures medial from the STN electrode could also interfere with premotor control of lid function. Perirubral fields were identified to be connected to the OO as revealed from tracer experiments in rats [START_REF] Morcuende | Neuronal premotor networks involved in eyelid responses: retrograde transneuronal tracing with rabies virus from the orbicularis oculi muscle in the rat[END_REF] and might be entrained easily by STN-DBS.

Interstitial nucleus of Cajal (InC), rostral intermedial longitudinal fascicle (riMLF), and the mgroup also participate in premotor control of the eyelid but are rather related to LPS function and upgaze than to tonic innervation of the OO [START_REF] Schmidtke | Nervous control of eyelid function. A review of clinical, experimental and pathological data[END_REF][START_REF] Horn | Neuroanatomical identification of mesencephalic premotor neurons coordinating eyelid with upgaze in the monkey and man[END_REF] .

IEC in PD5 and PD6 was completely reversed with dopaminergic medication or STN-DBS.

Overactivity of the OO was evident in these two patients and IEC presented with irregular (PD5) or tonic (PD6) contractions. Yet, responsiveness of IEC on levodopa, coincident contraction of frontalis muscle, and the ability to passively retract eyelids in PD5 provide evidence for possible LPS inhibition [START_REF] Reichel | Palpebral Blepharospasm -Distinction from Apraxia of Lid Opening and Disturbance of Inhibition by Synchronous Electromygraphy[END_REF][START_REF] Dewey | Isolated eyelid-opening apraxia: report of a new levodopa-responsive syndrome[END_REF][START_REF] Yamada | The effects of levodopa on apraxia of lid opening: A case report[END_REF][START_REF] Umemura | Apraxia of eyelid opening after subthalamic deep brain stimulation may be caused by reduction of levodopa[END_REF] or more likely disturbed antagonistic inhibition [START_REF] Reichel | Palpebral Blepharospasm -Distinction from Apraxia of Lid Opening and Disturbance of Inhibition by Synchronous Electromygraphy[END_REF][START_REF] Aramideh | Electromyographic features of levator palpebrae superioris and orbicularis oculi muscles in blepharospasm[END_REF][START_REF] Esteban | Eyelid movements in health and disease. The supranuclear impairment of the palpebral motility[END_REF] . Although no electrophysiological data were presented in previously published similar cases [START_REF] Dewey | Isolated eyelid-opening apraxia: report of a new levodopa-responsive syndrome[END_REF][START_REF] Yamada | The effects of levodopa on apraxia of lid opening: A case report[END_REF][START_REF] Umemura | Apraxia of eyelid opening after subthalamic deep brain stimulation may be caused by reduction of levodopa[END_REF][START_REF] Fuss | Improvement of apraxia of lid opening by STNstimulation in a 70-year-old patient with Parkinson's disease. A case report[END_REF][START_REF] Strecker | Increase of frequency in deep brain stimulation relieves apraxia of eyelid opening in patients with Parkinson's disease: case report[END_REF] , we think that the cases reported by these authors would be similar to those of PD5 and PD6 of our series because of their response to levodopa or stimulation.

Premotor control of lid-eye coordination by extrapyramidal and mesencephalic circuitries has been suggested and dopaminergic involvement was further substantiated by analysis of blinking rates in patients with PD and schizophrenia. Dyskinetic PD patients and untreated schizophrenic patients displayed increased blinking rates, whereas the opposite occurred in hypodopaminergic states [START_REF] Karson | Spontaneous eye-blink rates and dopaminergic systems[END_REF] . Excitability of reflex blinking was increased in dopaminergic off-state [START_REF] Agostino | Voluntary, spontaneous, and reflex blinking in Parkinson's disease[END_REF] and decreased after apomorphin administration [START_REF] Napolitano | Different effects of levodopa and apomorphine on blink reflex recovery cycle in essential blepharospasm[END_REF] . The present data add further evidence for nigro-striatal contribution to premotor control of lid-eye coordination, as stimulation effects on improvement of IEC already appeared at low stimulus intensities together with motor improvement. Side effects due to involvement of the capsula interna occurred with far higher stimulus intensities (table 2).

Alternatively, a stimulation-induced entrainment of premotor fibres of the central caudal oculomotor nucleus located in the vicinity of STN may be involved. As it innervates LPS and receives complex premotor afferents from basal ganglia including riMLF and InC, these fibres could be entrained in restauration of tonic LPS activity. However, the absence of oculomotor side effects and synergistic effects of levodopa argue in favor of effects mediated through nigrostriatal pathways.

In conclusion, IEC in PD after STN-DBS may occur as a direct side effect from stimulation and, therefore, respond to adequate stimulation programming, or as a direct correlate of the underlying neurodegeneration of dopaminergic neurons and, therefore, respond to STN-DBS or levodopa administration.

Systematic clinical and electromyographic characterisation may help in identifying the differences between the two forms of IEC seen after STN-DBS. In patients with IEC induced by STN-DBS, low intensity stimulation was necessary to avoid current spreading to neighbouring structures, which could also be avoided by bipolar stimulation. If no sufficient control of motor symptoms can be achieved without induction of IEC, botulinum toxin injections into the orbicular portion of the OO should be considered with generally good to excellent response.

Consideration of neuroanatomical correlates might argue in favor of a stimulus-related activation of corticobulbar fibres in the capsula interna in this group. This was further strengthened by the intraoperative observation that allowed an estimate of the localisation of this symptom.

A second group of patients showed IEC responsive to stimulation or levodopa intake, revealing an impairment of nigro-striatal premotor control of lid function. Co-contraction of frontalis muscle and the ability to passively retract eyelids were suggestive of concomitant inhibition of LPS or disturbed antagonistic inhibition. Our findings imply clues for therapeutic management of IEC after STN-DBS. Strategies for DBS programming and dopaminergic medical therapy can be diametrically opposed in regard to the pathophysiological mechanisms of IEC, and their appropriate allocation is necessary to achieve favorable clinical outcome.

Tables and figures

  beneficial for the control of motor symptoms in postoperative management and therapeutic efficacy of stimulation was documented by UPDRS III scores in PD5 (MedOff/StimOff: 42, MedOff/StimOn: 29) and PD6 (MedOff/StimOff: 57, MedOff/StimOn: 22). A further increase of amplitude on the respective contacts elicited dyskinesias and confirmed proper electrode localisation taken together with postoperative MRI.

  Abbreviations: CI = capsular interna; d = dysarthria; cf = contraction face; ca = contraction arm; p = paraesthesia; veg = vegetative; n = not present; nt = not tolerated; e = excluded from analysis

Table 1 : Clinical characteristics

 1

	patient	age	sex	H&Y	disease duration	uncontrolled motor symptoms	DBS-surgery	LED [mg]	UPDRS III
					[years]	prior to STN-DBS surgery	[month/year]	before/after STN-DBS	[StimOff/StimOn]
	PD1	59	male	5	26	peak-dose dyskinesias	02/2008	1450 / 970	43 / 16
						wearing off (end of dose)
						sudden offs

Clinical characteristics. Abbreviations: H&Y = Hoehn & Yahr stage before surgery. LED = levodopa equivalent dose in [mg]. *Patient seen in the operating room because of IEC induced by intraoperative test stimulation

Table 2 : Threshold amplitudes for induction or reversal of IEC and visible capsular side

 2

Table 3 : Results from clinical testing and analysis of OO-EMG

 3

	patient	onset of IEC after	effect of stimulation	effect of levodopa	OO-EMG pattern	root mean square [µV]
		STN-DBS [months]			StraightOFF / StraightON	StraightOFF / StraightON
	PD1	0	induction of IEC	nt	background / tonic overactivity	33 / 281
	PD2	6	induction of IEC	nt	background / tonic overactivity	291 / 1300
	PD3	12	induction of IEC	nt	background / tonic overactivity	214 / 711
	PD4	6	induction of IEC	nt	background/ tonic overactivity	82 / 670
	PD5	3	reversal of IEC	reversal of IEC	irregular overactivity / background	642 / 229

Funding

Licence for Publication

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and sublicences such use and exploit all subsidiary rights, as set out in our licence.

Competing interests

Dr. Asmus has received speakers honoraria and travel grants from Ipsen Pharma, Allergan and Merz Pharmaceuticals

Prof. Gasser has received speakers honoraria from Novartis, Merck-Serono, Schwarz Pharma, Boehringer Ingelheim, and Valeant Pharma and research grants from Helmholtz Alliance for Health in an Ageing Society (HELMA)


Douglass Cp

Kandler Rh

Shaw Pj

Mcdermott Cj

An evaluation of neurophysiological criteria used in the diagnosis of Motor Neurone Disease

Keywords:

New criteria for the neurophysiological diagnosis of Amyotrophic Lateral Sclerosis/ Motor Neurone Disease (ALS/MND) were recently proposed at an international symposium in Awaji-shima, Japan. They differ from the accepted revised El-Escorial criteria by considering fasciculation potentials to be evidence of acute denervation. In addition when assessing diagnostic certainty, the Awaji-shima criteria equate electrodiagnostic evidence of lower motor neurone dysfunction with clinical examination findings.

A retrospective review of 205 consecutive sets of notes was performed, from patients who underwent neurophysiological assessment for suspected MND. The clinical signs and neurophysiological findings were combined according to the two sets of criteria (revised El-Escorial and Awaji-shima) and the diagnoses reached were compared to the interval diagnosis, to establish the sensitivities and specificities of each protocol.

An interval diagnosis of MND was recorded in 107 patients. The sensitivity of the Awaji-shima criteria in reaching a diagnosis of MND was 60.7% and the revised El-Escorial 28%, with a specificity of 95.9% for both criteria. The Awaji-shima criteria increased the sensitivity of diagnosis without affecting the specificity.

Accepting EMG evidence of fasciculations as evidence of acute denervation increases the diagnostic certainty of MND and the new criteria allow earlier diagnosis of MND without increasing the false positive rate.

INTRODUCTION

Motor neurone disease (MND) is the third commonest adult onset neurodegenerative disorder, with an annual incidence of 2 per 100 000 and prevalence of 5-7 per 100 000. [START_REF] Mcdermott | Diagnosis and management of motor neurone disease[END_REF] MND leads to progressive muscle weakness and atrophy, with a mixture of upper and lower motor neurone signs, due to degeneration of pyramidal neurons in the motor cortex, cranial motor neurons and anterior horn cells in the spinal cord.

Classical MND tends to be focal in onset, with a particular group of motor neurones affected first. This can lead to a delay in diagnosis, as early in the disease course there may be few clinical signs. An earlier diagnosis reduces the period of diagnostic uncertainty for the patient and allows the timely planning of future care and support.

Additionally commencing neuroprotective agents, such as Riluzole, whilst there is a larger pool of surviving motor neurones may improve any therapeutic effect. [START_REF] Mcdermott | Diagnosis and management of motor neurone disease[END_REF] Neurophysiological evaluation is used in patients with suspected MND to both support the diagnosis, by finding evidence of a more widespread motor neurone dysfunction than is apparent clinically, and also to exclude other disorders which may mimic MND. Several sets of criteria have been proposed over the recent past, which combine the clinical evaluation and neurophysiological assessment to facilitate an earlier diagnosis of MND.

Lambert suggested that clinical neurophysiology could assist in the diagnosis of ALS and in the exclusion of other peripheral neuromuscular pathologies in two papers in 1956 and 1957 and later published a list of criteria for the electrophysiological confirmation of ALS in 1969. [START_REF] Lambert | Electromyography in amyotrophic Lateral Sclerosis[END_REF] In 1990 a three day workshop on "The clinical limits of ALS" was convened at El Escorial, Spain by the World Federation of Neurology Subcommittee on Motor Neuron Disease. Their aim was to develop diagnostic criteria which would include electrophysiological and clinical data, and be workable, internationally acceptable and provide an algorithm which would enhance clinical studies, therapeutic trials and molecular genetic research studies. [START_REF] Brooks | Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial Clinical limits of amyotrophic lateral sclerosis workshop contributors[END_REF] These criteria were revised in 1998 at a workshop in Airlie House, Warrenton, Virginia. [START_REF] Brooks | El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis[END_REF] The revised El Escorial/Airlie House criteria are summarised in Figure 1a. The body is divided into 4 regions, the brainstem, and the cervical, thoracic and lumbosacral spinal cord regions. The revised El Escorial criteria categorise the patients into 4 levels of diagnostic probability; Clinically definite ALS, Clinically probable, Clinically probable-laboratory supported (a category not present in the original El Escorial criteria), and Clinically possible ALS. The revised El Escorial criteria have been criticised for being too strict, resulting in some patients with clinically obvious MND never achieving a diagnostic category higher than Clinically Possible. One area for debate regarding the revised El-Escorial criteria has been the fact that fasciculations on electromyography (EMG) are not taken as evidence of lower motor neurone dysfunction. Lambert stated that "The EMG discloses fasciculations so regularly in ALS that one rarely accepts the diagnosis unless fasciculation is present". [START_REF] Lambert | Electromyography in amyotrophic Lateral Sclerosis[END_REF] The revised El Escorial criteria stated that fasciculation potentials were a characteristic clinical feature of ALS and that their absence raises diagnostic doubts, however they are not accepted as evidence of active denervation.

Wilbourn suggested that the El Escorial criteria should be revised within a less rigid framework so that fasciculations can be given the emphasis they merit. [START_REF] Wilbourn | Clinical neurophysiology in the diagnosis of amyotrophic lateral sclerosis: The Lambert and the El Escorial criteria[END_REF] In 2006 an international symposium was held in Awaji-shima, Japan to determine the best use and interpretation of electrophysiological data in the diagnosis of ALS. [START_REF] De Carvalho | Electrodiagnostic criteria for diagnosis of ALS. Consensus of an international Symposium sponsored by IFCN[END_REF] The Awaji-shima criteria proposed that electrophysiological and clinical signs of lower motor neurone (LMN) degeneration are given equal weight in the decision as to which diagnostic category the patient is assigned. The revised El Escorial criteria do not allow the EMG and clinical abnormalities to be combined in a single limb, a limb must be determined to be abnormal by one technique or the other.

The Awaji-shima criteria, by accepting neurogenic EMG abnormality as equivalent to clinical abnormality, allows a currently strong limb to be classified as abnormal earlier than if the decision were based on EMG or clinical criteria alone. [START_REF] De Carvalho | Electrodiagnostic criteria for diagnosis of ALS. Consensus of an international Symposium sponsored by IFCN[END_REF] This renders the clinically probable-laboratory supported ALS category redundant as all categories can now use laboratory support in the diagnosis.

In both sets of criteria EMG evidence of lower motor neurone involvement requires signs of both active and chronic denervation to be present. The presence of chronic denervation is confirmed by long duration, large amplitude, polyphasic, unstable motor unit potentials and decreased motor unit recruitment. Active denervation is confirmed by the presence of positive sharp waves and fibrillation potentials. These changes must be present in at least two muscles in the cervical and lumbosacral spinal cord regions and in one muscle in the brainstem and thoracic cord regions for the regions to be categorised as affected. The criterion for numbers of muscles involved in a region is shared by both the El Escorial and Awaji-shima groups and has been shown to achieve the best combination of sensitivity and specificity. [START_REF] Makki | The electromyographic diagnosis of amyotrophic lateral sclerosis: does the evidence support the El Escorial criteria?[END_REF] A further significant difference in the Awaji-shima criteria, is the reintroduction of fasciculation potentials as evidence of acute denervation, equivalent to that of fibrillation potentials and positive sharp waves. This is similar to the Lambert criteria and it has been suggested that these changes will increase the sensitivity of the diagnostic criteria for ALS without changing the specificity. [START_REF] Fuglsang-Friedericksen | Diagnostic criteria for amyotrophic lateral sclerosis (ALS)[END_REF] The El-Escorial criteria did not include fasciculations as part of the diagnostic criteria as they were considered to be less specific than fibrillations and could produce false positive results. The fasciculations that are accepted in the Awaji-shima criteria are not the simple and stable fasciculations that may occur in benign conditions but unstable, complex fasciculation potentials in the presence of unstable motor unit potentials in the context of suspected ALS.

AIMS

The aim of this study was to determine the sensitivity and specificity of the Awajishima criteria for diagnosing MND in a clinical/ non research setting as compared to the revised El Escorial criteria.

METHODS

The neurophysiology department database was interrogated to identify 250 consecutive patients, who had been referred from January 1 st 2000 with motor neurone disease included in the differential diagnosis. This time period was chosen as it would allow sufficient time for the interval diagnosis to become clinically evident. The clinical findings and neurophysiology results were then combined according to the two sets of criteria, and the diagnosis that would have been reached after the first clinical encounter and electro-diagnostic test, was recorded as the criteria diagnosis. The criteria diagnoses were then compared to the interval diagnoses and the sensitivity and specificity of the Awaji-shima and revised El-Escorial criteria were calculated. For the purpose of calculating the sensitivities and specificities, if the criteria diagnosis was clinically definite MND or clinically probable/ clinically probable-laboratory supported MND then these patients were grouped together as having a "positive MND criteria diagnosis". Conversely if the criteria diagnosis was clinically possible MND or the patient did not achieve a diagnostic category then these patient were assigned a "negative MND criteria diagnosis".

RESULTS

The clinical notes and the corresponding neurophysiology reports of 205 patients referred for EMG with suspected MND were reviewed. The interval diagnosis was MND in 107; of these 97 were limb onset amyotrophic lateral sclerosis and 10 were bulbar onset ALS. 3 patients had a family history of MND but were all SOD1 negative cases. The interval diagnosis in the remaining cases was varied. The largest groupings were neuropathies (22), radiculopathies (19), and benign cramp fasciculation syndrome [START_REF] De Carvalho | Electrodiagnostic criteria for diagnosis of ALS. Consensus of an international Symposium sponsored by IFCN[END_REF]. The remaining 51 had no evidence of neurological disease or a wide variety of disorders, with no suggestion of MND. The mean duration between initial clinical presentation and assessment, and assigning the interval diagnosis in this study was 89 months (range 65 to 116 months). The mean age of patients with MND was 64.3 (range 31 to 95). The mean duration of symptoms at the time of initial clinical presentation was 17.9 months (range 1 to 192 months) and mean time from clinical presentation to EMG was 1.2 months (range 0 to 37 months).

Mean length of follow up was 16.3 months (range 1 to 87 months). There was an approximate 3:1 male to female ratio. Using the numbers of patients with a positive or negative MND criteria diagnosis and the numbers of those patients who had a final interval clinical diagnosis of ALS or another diagnosis (table 1), we were able to calculate the sensitivity and specificity of the two sets of criteria. Using the clinical information of the upper and lower motor signs found on examination, interpreted using the revised El Escorial clinical criteria, to make the diagnosis of MND had a sensitivity of 27.1%. When information from the EMG was applied using the revised El Escorial criteria, the sensitivity rose to 28 %. Using the Awaji-shima criteria however increased the sensitivity to 60.7%. The specificity of each of the three methods was 95.9% indicating that there were no more false positives with the new criteria. [START_REF] Decarvalho | Awaji diagnostic algorithm increases sensitivity of El Escorial criteria for ALS diagnosis[END_REF] The sensitivity was increased to 95% using the Awaji-shima criteria compared to 53% when applying the revised El Escorial criteria. They studied 55 consecutive patients previously diagnosed with ALS by standard clinical techniques in whom neuropathies had been excluded by sensory and motor nerve conduction studies. They performed extensive EMG studies in at least 10 sites in each limb and cranially innervated muscle. Additionally, in 44 of the 55 patients the diaphragm and a trunk muscle were also studied. This level of investigation in a selected group of patients in a research setting may account for the higher sensitivity than we achieved in our less selected clinical group of patients. The specificity of the criteria was not investigated in their paper however and so our study is the first to demonstrate that the criteria are specific even in a clinical setting with standard neurophysiological testing. Using the Awaji-shima criteria facilitates an earlier diagnosis in patients with MND. This may have benefits in reducing periods of diagnostic uncertainty, reduce repetitive testing, enable earlier recruitment into clinical trials and commencement of supportive treatments and Riluzole.

DISCUSSION

LEGEND FOR FIGURES

Revised El Escorial criteria

Awaji shima critera

Diagnostic categories of patients with confirmed ALS as determined by the criteria.

TABLES Table 1

Numbers of patients assigned to each diagnostic category Table 2 Sensitivity and specificity of the diagnostic criteria.

  205 patients had full clinical records and EMG results available. The results of the electromyography and nerve conduction studies were re-examined and changes of chronic denervation and of active denervation (fibrillation potentials and positive sharp waves and/or complex fasciculations) in each area of the body were recorded.The nerve conduction studies were recorded as normal/ supportive of MND or the alternative diagnosis was recorded. All patients included in the study had a proximal and a distal muscle in an upper and lower limb sampled and a bulbar muscle was also examined where indicated. A sensory and motor nerve conduction study was performed in an upper and lower limb.The clinical notes were examined. The clinical signs on presentation were recorded as positive or negative for upper motor neurone and/or lower motor neurone signs in each area of the body. The clinical diagnosis from the last review was recorded as the interval diagnosis.

  figure 2.

  The Awaji-shima criteria have been shown to increase the sensitivity of diagnosis of MND compared to the revised El Escorial criteria, without an increase in the false positive rate. There were 4 patients who had a diagnosis of clinically probable ALS despite their final clinical diagnosis not being ALS. They gained this false positive diagnosis through the clinical signs (upper and lower motor neurone signs in 2 regions of the body due to degenerative spinal cord disease) rather than through the extra information gained from the neurophysiology testing. The Awaji-shima criteria would have categorised 35 more patients as having a positive diagnosis than the El Escorial criteria (69 vs 34 had a diagnosis of clinically definite or probable). These 35 patients could have been entered into clinical trials at this point and neurologists investigating these patients may have felt more confident in giving a definite diagnosis to the patients and initiating Riluzole and supportive interventions. In January 2009 De Cavalho and Swash suggested that the Awaji-shima algorithm superimposed onto the El Escorial criteria in a research setting created an increased sensitivity of diagnosis.

  Figure 1b.

Table 2 .

 2 Sensitivity and specificity of the diagnostic criteria.

		Sensitivity	Specificity
	Pre-EMG diagnosis	27.1%	95.9%
	El Escorial criteria	28.0%	95.9%
	Awaji Shima criteria	60.7%	95.9%

Acknowledgements

The authors would like to thank Drs John Jarratt and Arup Chattopadhyay for the neurophysiological testing of patients, the Sheffield neurologists for allowing access to the notes of their patients and their secretaries for assistance in obtaining the notes.

Funding.

None.

The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non-exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd, and its Licensees to permit this article (if accepted) to be published in JNNP and any other BMJPGL products and to exploit all subsidiary rights, as set out in our licence.

Competing interests.

None.

ALS + ALS-