MD Susanne Koch
email: susanne.koch@charite.de

Simone Spuler

Maria Deja

Jeffrey Bierbrauer

Anna Dimroth

Friedrich Behse

Claudia D Spies

Klaus-D Wernecke

Steffen Weber-Carstens

Critical illness myopathy is frequent -accompanying neuropathy protracts ICU discharge

Keywords: critical illness myopathy, critical illness polyneuropathy, direct muscle stimulation, intensive care unit CIM = Critical Illness Myopathy, CIP = Critical Illness Polyneuropathy, dmCMAP = Compound Muscle Action Potential after direct muscle stimulation, ICU = Intensive Care Unit, MFCV = Muscle Fibre Conduction Velocity, MUAP

Objectives: Neuromuscular dysfunction in critically ill patients is attributed to either critical illness myopathy (CIM) or critical illness polyneuropathy (CIP) or a combination of both. But it is unknown whether differential diagnosis has an impact on prognosis. This study investigates whether there is an association between the early differentiation of CIM versus CIP and clinical prognosis.

Methods: We included mechanically ventilated patients who featured a Simplified Acute Physiology Score II (SAPS-II) ≥ 20 on three consecutive days within the first week after ICU admission. 53 critically ill patients were enrolled and examined by conventional nerve conduction studies and direct muscle stimulation (184 examinations in total). The first examination was conducted within the first week after admission to the intensive care unit (ICU).

Results: In this cohort of critically ill patients, CIM was more frequent (68%) than CIP (38%).

Electrophysiological signs of CIM preceded electrophysiological signs of CIP (median at day 7 in CIM patients vs. day 10 in CIP patients, p<0.001). Most patients with CIP featured concomitant CIM. At discharge from ICU, 25% of patients with isolated CIM showed electrophysiological signs of recovery and significantly lower degrees of weakness. Recovery could not be observed in patients with combined CIM/CIP even though ICU length of stay was significantly longer (mean 35 days in CIM/CIP vs. mean 19 days in CIM, p<0.001).

Conclusion:

Prognoses of patients differ depending on electrophysiological findings during early critical illness: Early electrophysiological differentiation of ICU acquired neuromuscular disorder enhances the evaluation of clinical prognosis during critical illness.

Introduction

Since the early description of critical illness myopathy (CIM) [START_REF] Macfarlane | Severe myopathy after status asthmaticus[END_REF] and critical illness polyneuropathy (CIP) [START_REF]Disseminated neuropathy following coma. On the differentation of so-called toxic polyneuropathy[END_REF][START_REF] Bolton | Polyneuropathy in critically ill patients[END_REF], the primary cause of these Intensive Care Unit (ICU) acquired weaknesses remains unresolved. [START_REF] Schweickert | ICU Acquired weakness[END_REF] ICU acquired weakness complicates recovery in critically ill patients and prolongs duration of mechanical ventilation and length of stay in ICU. [START_REF] De Jonghe | Paresis acquired in the intensive care unit[END_REF][START_REF] Garnacho-Montero | Critical illness polyneuropathy: risk factors and clinical consequences. A cohort study in septic patients[END_REF] Some authors advise against differentiation of CIM and CIP, as differentiation is complicated and may not result in consequences. [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Hermans | Impact of intensive insulin therapy on neuromuscular complications and ventilator dependency in the medical intensive care unit[END_REF] Electrophysiological abnormalities such as low compound muscle action potentials or pathological spontaneous activity can be detected within one week of ICU admission but do not allow for distinguishing CIM and CIP. [START_REF] Tepper | Incidence and onset of critical illness polyneuropathy in patients with septic shock[END_REF][START_REF] Tennilä | Early signs of critical illness polyneuropathy in ICU patients with systemic inflammatory response syndrome or sepsis[END_REF] In sedated patients without voluntary muscle contraction electrophysiological differentiation between CIM and CIP is difficult. CIM diagnosis relies on direct demonstration of muscle membrane dysfunction. [START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Lefaucheur | Origin of ICU acquired paresis determined by direct muscle stimulation[END_REF] Authors using this technique of direct muscle stimulation found myopathy to be more frequent in critically ill patients. [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Lefaucheur | Origin of ICU acquired paresis determined by direct muscle stimulation[END_REF][START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF][START_REF] Allen | Critical illness myopathy: Further evidence from muscle-fiber excitability studies of an acquired channelopathy[END_REF][START_REF] Weber-Carstens | Non-excitable muscle membrane predicts ICUacquired paresis in mechanically ventilated, sedated patients[END_REF] CIP with associated sensory nerve involvement is assessed by a reduction of sensory nerve amplitudes, whereas assessment of motor CIP remains difficult.

Rich and colleagues introduced a ratio that divides nerve evoked compound muscle action potentials (neCMAP) by direct muscle evoked compound muscle action potentials (dmCMAP) in order to differentiate between myopathy (this ratio is expected to be near 1) and motor CIP axonopathy (this ratio is expected to be small and near zero). [START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF] In this longitudinal study of 53 critically ill patients, conventional nerve conduction studies and direct muscle stimulation were combined to determine whether there is an association between the early differentiation of CIM versus CIP and clinical prognosis.

Methods

The study was approved by our local ethics committee. Written informed consent of legal proxies was obtained according to the Declaration of Helsinki.

We performed this observational study in a 14-bed surgical ICU over a period of 18 months.

Patients who required mechanical ventilation and featured a Simplified Acute Physiology

Score II > 20 (SAPS-II) [START_REF] Gall | A new Simplified Acute Physiology Score (SAPS-II) based on a European/North American multicenter study[END_REF] on three consecutive days within the first 7 days after ICU admission were eligible for inclusion. Pre-existing neuromuscular disorders, severe head trauma or bleeding diathesis were previously defined as exclusion criteria. All patients were treated according to our standard operating procedures of intensive care medicine, adopting evidence based bundles for severe sepsis. [START_REF] Deja | Evidence-based therapy of severe acute respiratory distress syndrome: an algorithm-guided approach[END_REF]19] Severity of illness was monitored daily by repeated ratings of SAPS-II and Sequential Organ Failure Assessment score (SOFA). [START_REF] Vincent | The SOFA (sepsis-related organ failure assessment) score to describe organ dysfunction/failure. On behalf of the working group on sepsisrelated problems of the european society of intensive care medicine[END_REF] Every three days after admission to the ICU electrophysiological bedside studies were recorded by portable 2-Channel Keypoint Medtronic equipment (Skovlunde, Denmark). Once either CIM or CIP was detected or once patients showed adequate awareness, diagnostic testing was repeated once a week. Given that patients featured sufficient awareness at ICU discharge (Ramsay score < 2) [START_REF] Ramsay | Controlled sedation with Alphaxalonealphadolone[END_REF], muscle strength of upper and lower limbs was evaluated and graded according to the Medical Research Council score. [START_REF]Aids to the examination of the peripheral nervous system[END_REF] Whenever possible, we examined two proximal/distal muscles in each extremity and divided the total by the number of muscles examined.

Motor nerve conduction velocity and compound muscle action potential amplitude after nerve stimulation (neCMAP) were unilaterally performed in median, peroneal as well as in tibial nerves and recorded from abductor pollicis brevis, extensor digitorum brevis and abductor hallucis muscles. Sensory nerve conduction studies were unilaterally conducted in sural and median nerves. Surface electrodes were used for stimulation and recording. In case of missing recordings or in presence of oedema, subdermal electrodes were used in all patients. Nerve conduction measurements were compared to normal values from age-matched individuals that were provided by the neurophysiological laboratory of the Charité.

During sedation, electromyography was performed in deltoid, biceps brachii, extensor digitorum longus, abductor pollicis brevis, rectus femoris, and tibialis anterior muscles using concentric needle electrodes to assess pathological spontaneous activity. As soon as patients showed sufficient awareness and voluntary muscle contraction was possible, quantitative electromyography was applied whenever possible in extensor digitorum longus and tibialis anterior muscles. A total of 20 different motor unit action potentials (MUAP) was sampled by random insertion of a concentric needle electrode into four different regions of an examined muscle, each recorded at 10ms, 50µV and filter settings of 500 Hz and 10 kHz. [START_REF] Trojaborg | Quantitative electromyography in polymyositis: a reappraisal[END_REF][START_REF] Buchthal | The diagnostic yield of quantified electromyography and quantified muscle biopsy in neuromuscular disorders[END_REF] Mean duration of collected non-polyphasic MUAPs was compared to normal values from healthy age-matched volunteers. [START_REF] Trojaborg | Quantitative electromyography in polymyositis: a reappraisal[END_REF][START_REF] Rosenfalck | Electromyography, sensory and motor conduction: findings in normal subjects[END_REF] Assessment of compound muscle action potential amplitudes following direct muscle stimulation (dmCMAP) was performed by longitudinal placement of either conventional stimulating surface electrodes or by subdermal electrodes along muscle fibres just proximal of the distal tendon insertion in case of oedema. Muscles were stimulated by gradually increasing strength (from 10 to 100mA) at 1Hz and a pulse duration of 0.1ms. For recordings, disposable concentric needle electrodes (length 25mm or 37mm; diameter 0,46mm) and/or disposable gel surface electrodes were used and placed 15-50 mm proximal of the stimulating electrode, guided by muscle twitch. Whenever no twitch was visible, the recording concentric needle electrode was pointed at four different directions in order not to miss small amplitudes.

Muscles were assumed to be inexcitable if responses could still not be obtained. dmCMAP amplitudes were measured peak to peak. Filter settings were 500 Hz and 10 kHz. Limbs' temperature was ensured to be >32°C. The examination included tibialis anterior and abductor pollicis brevis. According to Trojaborg and colleagues, dmCMAP amplitudes recorded by concentric needle electrodes <3mV were considered to be pathologic and consistent with myopathy. [START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF] Electrophysiological measurements of tibialis anterior and abductor pollicis brevis muscles of healthy volunteers (age 22 -74 years) in our laboratory provided reference values for dmCMAP amplitudes using surface electrodes (n=17) or concentric needle electrodes (n=8).

To assess motor CIP we calculated neCMAP/dmCMAP ratios (recorded in tibialis anterior muscle with peroneal nerve stimulation at the knee AND in abductor pollicis brevis muscle with median nerve stimulation at the wrist) as introduced by Rich and colleagues: ratios <0.5 indicate motor neuropathy; ratios >0.5 in combination with reduced dmCMAP amplitudes indicate myopathy, while ratios >0.5 in presence of normal dmCMAP amplitudes display normal findings. [START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF] To determine muscle-fibre conduction velocity (MFCV), latencies of muscle-fibre action potentials were determined and calculated for the measured distance between electrodes. Perpendicular position of recording needle electrodes was ensured. Responses earlier than 8 ms were likely to be conducted via intramuscular nerve twigs and not included. [START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Lefaucheur | Origin of ICU acquired paresis determined by direct muscle stimulation[END_REF][START_REF] Allen | Critical illness myopathy: Further evidence from muscle-fiber excitability studies of an acquired channelopathy[END_REF] Diagnostic criteria for electrophysiological examination were as follows:

ICU control: patients presenting no pathology; ICU unspecific: patients presenting unspecific pathology (pathological spontaneous activity and reduced neCMAP) not verifying myopathy or neuropathy; CIM patients: patients presenting reduced dmCMAP in at least one muscle examined in addition to unspecific findings and normal sensory/motor nerve conduction velocity (isolated CIM); CIP patients: patients presenting reduced SNAP and/or ne/dmCMAP ratio <0.5 in addition to unspecific findings (isolated CIP), CIM/CIP patients: patients presenting characteristics of combined CIM and CIP -reduced dmCMAP AND reduced SNAP and/or ne/dmCMAP ratio <0.5.

Patients were classified according to their most severe electrophysiological findings during their ICU stay. To compare electrophysiological data between patient groups, findings of the first examination presenting the most severe electrophysiological classification were chosen for each patient.

Results are expressed as arithmetic mean ± standard deviation (SD) for electrophysiological data, median and (25/75) percentiles (if number of patients <4 only median) for categorical or non-normally distributed data, or frequencies [%] for qualitative data, respectively. Statistical tests were conducted with non-parametric tests by Mann-Whitney-U test for two independent samples, Kruskal-Wallis test for three or more independent samples, and Fisher's exact test for qualitative data. In case of small samples, greater differences in sample sizes, large but unbalanced groups, data sets containing ties, or sparse data, tests were carried out in an exact version. Diagnostic test performance was evaluated by receiver operating characteristics (ROC) analysis using MUAP duration in tibialis anterior muscle <11.1 ms as electrophysiological gold standard for diagnosing myopathy in patients capable of voluntary muscle contraction or, alternatively, using amplitudes of dmCMAP in tibialis anterior muscles of sedated patients not being capable of voluntary muscle contraction.

Kaplan-Meier curves were estimated to show the cumulative incidence of different electrophysiological disorders developing over time and to estimate probabilities for ICU discharge after first day of awareness in CIM and CIM/CIP patients. Differences between groups considering cumulative incidences were tested by univariate Log-Rank test.

In univariate Cox' proportional hazard regressions we tested the impact of CIM respectively CIM/CIP as well as illness severity on the duration between first day of adequate awareness and ICU discharge (as dependent variable). In Cox' regressions with time dependent covariates, dmCMAP and SNAP amplitudes were included as indicators of myopathy respectively neuropathy while repeated recordings of SAPS-II and SOFA score during adequate awareness were included as indicators of illness severity. These variables were also analysed by stepwise (backward) procedure of multivariate Cox' regression accounting for time dependent covariates. Hazard ratios (HR) with 95%-confidence intervals (95%-CI (HR)) and corresponding p-values were calculated for each risk factor. p<0.05 (two-sided) was considered as statistically significant. Statistical analysis was performed using SPSS, Version

Results

212 patients required mechanical ventilation and featured SAPS II > 20 on three consecutive days within the first 7 days after ICU admission and were therefore eligible for inclusion.

Patients with pre-existing neuromuscular disorder (n=24), severe head trauma (n=14) or bleeding diathesis (n=33) (thrombocytopenia <20.000/µl) were excluded. 16 patients could not be included due to logistical reasons. Written informed consent by legal proxy could not be obtained in 72 patients. Finally, 53 patients were included and a total of 184 electrophysiological examinations were conducted. One or two examinations were conducted in nine patients each, three exams in 12 patients and four or more examinations in 23 patients.

Patient classification is shown in Figure 1. Only one patient with pre-existing Wilson's disease and two previous liver transplantations showed reduced SNAP amplitudes without evidence of motor CIP or CIM at both exams. However, pre-existing sensory polyneuropathy could not be ruled out. [START_REF] Leven | Lesions of the peripheral nerves in Wilson's disease. Electrodiagnostic findings[END_REF] To prevent confusion, all three patients classified as ICU unspecific as well as the only patient with isolated sensory nerve involvement were not considered in tables and figures.

Patients' characteristics are shown in Table 1. Amplitudes of nerve conduction studies are shown in Table 2. Predominant involvement of the lower limbs was observed in all patients. Parameters such as motor/sensory nerve conduction velocity, distal motor latency or F-Wave did not deviate from normal values in any of the examined patients. Pathological spontaneous activity such as fibrillation potentials or positive sharp waves were mostly of moderate activity and could be observed in different muscles from patients classified as "ICU unspecific", "CIM" and "CIM/CIP". Tibialis anterior and extensor digitorum longus muscles were most frequently affected. Muscle specific electrophysiological data are shown in Table 3. In quantitative electromyography we observed an increased incidence of polyphasic potentials and the recruitment pattern at maximum effort was fully or only mildly reduced despite severe weakness in patients classified as "CIM / CIP" respectively "CIM" indicating a myopathy.

We did not find signs of denervation such as a reduced recruitment pattern or elevated MUAP amplitudes.

In healthy subjects (age 22 -74 years) dmCMAP amplitudes were not below 0.6mV when recorded by surface electrodes and not below 3mV when recorded by concentric needle electrodes. For surface electrodes 95% CI were between 0.9mV and 6mV for tibialis anterior muscle and between 0.6mV and 20mV for abductor pollicis brevis muscle. Considering concentric needle electrodes, the according 95% CI were between 4mV and 19mV and between 6.8mV and 13mV, respectively.

MFCV was positively correlated with dmCMAP amplitude, reduced amplitudes indicating slower MFCV (Rho = 0.55 and R Quadrate = 0.401).

We did not observe isolated motor CIP in any patient, as ne/dmCMAP ratios were consistently >0.5, which indicates either myopathy in presence of reduced dmCMAP amplitudes (CIM-and CIM/CIP patients) OR normal findings in presence of normal dmCMAP amplitudes (healthy subjects and ICU-controls). ROC analysis verified dmCMAP in tibialis anterior muscle during sedation as predictor of myopathy as later diagnosed by MUAP duration in the same muscle once voluntary contraction was applicable. The best relationship of sensitivity (70%) to specificity (83.3%) was observed at the cut-off value of 3.2mV for dmCMAP, which is compatible to standard values from Trojaborg and colleagues. [START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF] Onset of electrophysiological pathology is shown in Figure 2. Six patients could not be included in analysis due to treatment in external hospitals prior to ICU admission. Abnormal dmCMAP amplitudes occured significantly earlier than abnormal SNAP (p < 0.001), indicating that CIM occurs prior to CIP with associated sensory nerve involvement during early critical illness. CIM patients showed reduced dmCMAP amplitudes median at day 7 (5/11) while CIM/CIP patients showed reduced SNAP amplitudes median at day 10 (4/13).

Confounders prolonging ICU length of stay showed that classification as CIM or CIM/CIP independently influenced ICU length of stay whereas illness severity was comparable between both groups (p=0.005) (Table 4).

Once sedation was ended, patients classified as CIM/CIP stayed significantly longer than patients classified as CIM (p = 0.05) (Figure 3). At discharge from ICU, some patients classified as CIM featured amplitude recovery of dmCMAP in tibialis anterior muscles (Figure 4a) and neCMAP in tibialis and peroneal nerves, while patients classified as CIM/CIP consistently showed reduced amplitudes of dmCMAP and neCMAP (Figure 4b).

At ICU discharge, muscle strength according to the MRC-score was significantly lower in patients classified as CIM/CIP (n=14; examination was precluded in 4 patients due to death and in 2 patients due to logisitical reasons; mean MRC-score in upper limbs 3.5; mean MRC-score in lower limbs 3.25) than in patients classified as CIM (n=8; examination was precluded in 6 patients due to death and in 2 patients due to logistical reasons; mean MRC-score in upper limbs 4.5, p=0.002; mean MRC-score in lower limbs 4.0, p=0.004).

Discussion

Direct muscle stimulation facilitates diagnosis of CIM in the early course of critical illness.

During analgesia and sedation, other methods of clinical assessment are not applicable.

Electrophysiological signs of CIM precede electrophysiological signs of CIP. Isolated CIP was not observed in any patient, it occurred only in combination with myopathy. Clinical courses of patients classified as CIM respectively CIM/CIP differ. Both CIM and CIM/CIP independently influence ICU length of stay after the end of sedation. However, patients classified as CIM/CIP feature significantly higher degrees of weakness at ICU discharge and longer ICU lengths of stay than patients classified as CIM. Electrophysiological recordings displayed that some patients classified as CIM showed signs of recovery at discharge from ICU while all patients classified as CIM/CIP consistently featured electrophysiological pathology at ICU discharge.

Technical aspects

By comparing dmCMAP amplitudes with MUAP duration in quantitative electromyographythe gold standard of proving myopathy [START_REF] Trojaborg | Quantitative electromyography in polymyositis: a reappraisal[END_REF][START_REF] Buchthal | The diagnostic yield of quantified electromyography and quantified muscle biopsy in neuromuscular disorders[END_REF] -we could show that assessment of dmCMAP amplitudes represents a valuable tool to differentiate between CIM and CIP during the early course of critical illness, when voluntary muscle contraction is not applicable due to sedation (sensitivity 70%, specificity 83.3%).

The technique of direct muscle stimulation has been evaluated in healthy subjects [START_REF] Troni | Conduction velocity along human muscle fibers in situ[END_REF][START_REF] Zwarts | Evaluation of the estimation of muscle fiber conduction velocity. Surface versus needle method[END_REF] and patients suffering from weakness and/or weaning failure caused by critical illness. [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Lefaucheur | Origin of ICU acquired paresis determined by direct muscle stimulation[END_REF][START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF][START_REF] Rich | Muscle is electrically inexcitable in acute quadriplegic myopathy[END_REF] Published reference data for dmCMAP depend on recording characteristics of electrodes: concentric needle electrodes (Trojaborg et of muscle membrane excitability on a subclinical level that is not accompanied by distinct levels of weakness after the end of sedation. [START_REF] Weber-Carstens | Non-excitable muscle membrane predicts ICUacquired paresis in mechanically ventilated, sedated patients[END_REF] This indicates, that critical illness in general causes impairment of muscle membrane excitability, however, in order to cause muscle organ failure an additional pathomechanism is essential.

Ne/dmCMAP ratios <0.5 are supposed to indicate motor axonopathy. [START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF] Since this ratio was >0.5 in all of our patients the presence of motor axonopathy is questionable. Z'Graggen and colleagues assessed the existence of membrane depolarization in motor nerves by applying nerve excitability testing [START_REF] Z'graggen | Nerve excitalility changes in critical illness polyneuropathy[END_REF], proving a nerve membrane affection but not finally proving the existence of a motor axonopathy.

It should be emphasised that SNAP abnormalities indicate sensory neuropathy and cannot serve as definite evidence towards neuropathic involvement in clinical weakness. Early reports attributed weakness in critically ill patients mostly to distal motor axonopathy on the basis of non-specific electrophysiological abnormalities and neglected the possibility of primary muscle fibre disorder. [START_REF] Bolton | Polyneuropathy in critically ill patients[END_REF][START_REF] Zochodne | Critical illness polyneuropathy. A complication of sepsis and multiple organ failure[END_REF] By applying the technique of direct muscle stimulation, we and others, were able to show that CIM is frequent in critically ill patients. [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Lefaucheur | Origin of ICU acquired paresis determined by direct muscle stimulation[END_REF][START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF][START_REF] Allen | Critical illness myopathy: Further evidence from muscle-fiber excitability studies of an acquired channelopathy[END_REF][START_REF] Weber-Carstens | Non-excitable muscle membrane predicts ICUacquired paresis in mechanically ventilated, sedated patients[END_REF] However, ne/dm CMAP ratios did not add further information to the diagnosis of myopathy than dmCMAP amplitudes.

Clinical aspects

Onset and incidence of neuromuscular disorder

To the best of our knowledge, this is the first study reporting that CIM is verified significantly earlier than CIP. Unspecific findings such as pathological spontaneous activity or reduced neCMAP amplitudes do not differentiate CIM and CIP and were observed within the first week after ICU admission. This is in line with three earlier studies describing early onset of neuromuscular dysfunction in the ICU without differentiating between myopathy and neuropathy. [START_REF] Tepper | Incidence and onset of critical illness polyneuropathy in patients with septic shock[END_REF][START_REF] Tennilä | Early signs of critical illness polyneuropathy in ICU patients with systemic inflammatory response syndrome or sepsis[END_REF][START_REF] Latronico | Simplified electrophysiological evaluation of peripheral nerves in critically ill patients: the Italian multi centre CRMYNE study[END_REF] We presume that early pathological spontaneous activity is related to muscle membrane depolarization leading to elevated excitability.

Coexistance of pathological spontaneous activity and reduced dmCMAP amplitudes was surprising to us, as we expected a reverse relationship due to contrary pathology. However, this is in line with findings from a rat-model of CIM describing concomitant membrane depolarization and reduced excitability, which was attributed to voltage-gated sodium-channel dysfunction. [START_REF] Rich | Crucial role of sodium channel fast inactivation in muscle fibre inexcitability in a rat model of critical illness myopathy[END_REF][START_REF] Filatov | Hyperpolarized shifts in the voltage dependence of fast inactivation of Na v 1,4 und Na v 1,5 in a rat model of critical illness myopathy[END_REF] It was furthermore reported, that endotoxin of gram-negative bacteria causes a hyperpolarized shift in the gating of voltage-gated sodium channels in human skeletal muscle, [START_REF] Haeseler | Endotoxin reduces availability of voltage-gated human skeletal muscle sodium channels at depolarized membrane potentials[END_REF] 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 Group and co-owners or contracting owning societies (where published by the BMJ Group on their behalf), and its Licensees to permit this article (if accepted) to be published in Journal of Neurology, Neurosurgery and Psychiatry with practical Neurology and any other BMJ Group products and to exploit all subsidiary rights, as set out in our licence.

Legends for figures

  compares all three groups (Kruskal-Wallis-test / Fisher's exact test), whereas pvalue b (Mann-Whitney-U test / Fisher's exact test) compares data between CIM-and CIM/CIP-patients. Values are shown as median and (25/75 percentiles) or as absolute numbers. BMI = Body Mass Index, ARDS = Acute Respiratory Distress Syndrome, SAPS-II = Simplified Acute Physiology Score, SOFA = Sepsis related Organ Failure Assessment, ICU length of stay = Intensive Care Unit length of stay.

  compares all three groups (Kruskal-Wallis-test), whereas p-value b (Mann-Whitney-U test) compares data between CIM-and CIM/CIP-patients. Values are given as mean ± SD. neCMAP = nerve evoked Compound Muscle Action Potential amplitude, SNAP = Sensory Nerve Action Potential amplitude.

  p-value b (Mann-Whitney-U test) compares data between CIM-and CIM/CIP-patients. Values are given as mean ± SD. dmCMAP = direct muscle stimulated Compound Muscle Action Potential amplitude, MFCV = Muscle Fibre Conduction Velocity, ne/dmCMAP ratio = nerve evoked compound action potential amplitude divided by direct muscle stimulated compound action potential amplitude, MUAP = Motor Unit Action Potential, Tib ant = tibialis anterior muscle, Abd poll brev = abductor pollicis brevis muscle, Ext dig com = extensor digitorum communis muscle.

  sedation and ICU discharge (dependent variable) is shown. Analyses included dmCMAP and SNAP amplitudes as indicators of CIM and/or CIP, respectively as well as repeated SAPS-II and SOFA score ratings during awareness as indicators of critical illness severity. Hazard ratios (HR) with 95%-confidence intervals (95%-CI[HR]) and p-values for each variable. SAPS-II = Simplified Acute Physiology Score, SOFA = Sepsis related Organ Failure Assessment, ICU = Intensive Care Unit, dmCMAP = direct muscle stimulated Compound Muscle Action Potential amplitude, SNAP = Sensory Nerve Action Potential amplitude.

Figure 1

 1 Figure 1 Consort Diagram for electrophysiological characteristic. SAPS-II = Simplified acute

Figure 2

 2 Figure 2 Cumulative incidence of neuromuscular affection in days after onset of critical

Figure 3

 3 Figure 3 Cumulative Probability for ICU length of stay counted from the day after awakening

Figure 4

 4 Figure 4 Time course for dmCMAP after onset till discharge of ICU stay organized in time

  14, Copyright© SPSS, Inc., Chicago, Illinois 60606, USA, and SAS, Version 9.1, Copyright© SAS Institute, Inc., Cary, NC, USA.

Table 1 Clinical characteristics of ICU-control, CIM-and CIM/CIP-patients

 1

		ICU -control (n=13)	CIM (n=16)	CIM/CIP (n=20)	p-value	a	p-value	b
	age (years)	34 (23/51)	59 (37/67)	51 (41/58)	0.063		0.301
	gender (m / f)	10/3	8/8	17/3	0.061		0.034
	BMI kg/m² on admission	24.7 (21.6/26.3)	25 (22/28.5)	26 (21.9/31.2)	0.445		0.404
	Sepsis on admission (%)	0%	44%	55%	0.002		0.738
	Diagnosis on admission
		76.9%	37.5%	50%	0.017		0.047
	Multiple trauma	(n=10)	(n=6)	(n=10)
	Cancer surgery	7.7%	37.5%	5%
		(n=1)	(n=6)	(n=1)
	ARDS / sepsis	7.7%	25%	45%
		(n=1)	(n=4)	(n=9)
	others	7.7%	-	-
		(n=1)
	SAPS-II on admission	26 (22/40)	39 (31/48)	41 (33/50)	0.227		0.539
	SAPS-II peak (first 10 days)	36 (24/44)	40 (31/52)	54 (40/65)	0.048		0.243
	SOFA on admission	8 (6/9)	9.5 (7/12)	11 (8/14)	0.006		0.149
	SOFA peak (first 10 days)	8 (7/9)	10 (8/14)	12 (10/14)	0.002		0.523
	Acute renal failure	0%	18.8%	25%	0.157		0.709
	on admission
	survival (%)	100%	62.5%	80%	0.045		0.285
	ICU length of stay (days)	8 (7/15)	19 (14./33)	35 (23/47)	<0.001	0.004
	p-value a

Table 2 Motor and sensory amplitudes for ICU control, CIM -and CIM/CIP patients.

 2

		Nerve	ICU -control (n=13)	CIM (n=16)	CIM/CIP (n=20)	p-value	a	p-value	b
	neCMAP (mV)	Median	10.1± 4.3	6 ± 4.5	4.5 ± 2.9	0.003		0.51
			(n=11)	(n=11)	(n=15)
		Peroneal	5.3 ± 3.7	2.3 ± 3.4	1 ± 1.2	0.003		0.32
			(n=13)	(n=15)	(n=19)
		Tibial	11.8 ± 6.1	3.2 ± 4.1	3.7 ± 4.2	0.013		0.16
			(n=13)	(n=15)	(n=13)
	SNAP (µV)	Median	22.1 ± 7.9	17.3 ± 6.7	15.4 ± 7.6	0.058		0.4
			(n=11)	(n=11)	(n=17)
		Sural	6.5 ± 3.4 (n=12)	12.8 ± 21.8 (n=15)	(n=18) 1.5 ± 2	<0.001	<0.001
	p-value a

Table 3 Muscle specific data for healthy volunteers, ICU control-, CIM-and CIM/CIP patients

 3

		muscle	healthy volunteers (n=17)	ICU controls (n=13)	(n=16) CIM	(n=20) CIM/CIP	p-value	a p-value	b
	dm CMAP (mV)	Tib ant	8 ± 2.1	5.6 ± 1.8	2.2 ± 2.3	1.6 ± 1.9	<0.001	0.008
	(concentric needle		(n=8)	(n=12)	(n=15)	(n=19)
	electrode)
	lower limit =3mV
		Abd poll brev	9.3 ± 2.3	4.3	4.2	2.6 ± 5	0.136		0.006
			(n=6)	(n=2)	(n=2)	(n=10)
	dm CMAP (mV) (surface electrode) lower limit =0.6mV	Tib ant	2.8 ± 0.4 (n=17)	(n=9) 1.6 ± 0.5	(n=9) 0.3 ± 0.1	(n=8) 0.1 ± 0.1	0.010		0.139
		Abd poll brev	7.1 ± 5.3 (n=12)	4.5 ± 4.1 (n=5)	1.3 ± 1.7 (n=6)	0.7 ± 1.6 (n=6)	0.044		0.134
	MFCV (m/s)	Tib ant	-	5.9 ± 1.6	5.2 ± 1.1	4.6 ± 1.1	0.454		0.335
				(n=3)	(n=8)	(n=9)
	ne/dmCMAP ratio	Tib ant	1.79 ± 1.02 (n=7)	2.2 ± 0.7 (n=9)	(n=4) 13.8 ± 14.6	(n=9) 4.9 ± 3.9	0.018		0.276
	MUAP duration (ms)	Tib ant	-	11.2 ± 0.6 (n=8)	9.0 ± 2.6 (n=7)	6.9 ± 1.8 (n=10)	0.001		0.477
		Ext dig com	-	10 ± 1 (n=8)	6.3 ± 3.1 (n=9)	5.6 ± 1.1 (n=12)	0.001		0.246
	p-value a compares ICU-controls, CIM-and CIM/CIP-patients (Kruskal-Wallis-test), whereas

Table 4 Confounders prolonging ICU length of stay between end of sedation and ICU discharge Univariate Cox' regression with time dependent covariates

 4

	Hazard Ratio[HR] 95%-CI[HR]	p

Univariate and multivariate Cox' proportional hazard regression accounting for time dependent covariates as potential confounders prolonging ICU length of stay between end of

  which in presence of muscle membrane depolarization in critically ill patients[36] will finally cause muscle membrane inexcitability. CIP was associated with more severe weakness at ICU discharge and longer ICU length of stay than isolated CIM. During the early course of critical illness, when voluntary muscle contraction is not applicable due to sedation, we recommend conventional

	electrophysiological recordings in combination with direct muscle stimulation (adds another
	5-15 min) to maintain precise differential diagnosis. This supports better prediction of
	weaning difficulties which occur in both CIM and CIM/CIP patients and furthermore assists
	clinicians in estimating motor function recovery at ICU discharge.
	We observed both systemic inflammation and illness severity during early critical illness to

present significant risk factors for development of CIM or CIM/CIP. It nevertheless remains unresolved why some patients show more severe or combined affection of muscles and nerves than others. with Acknowledgment This study was supported by the Deutsche Forschungsgemeinschaft (KFO 192, WE 4368/ 1-1). Competing Interest: None declared. The authors want to thank Prof. Dr. Werner Trojaborg for advice and assistance in direct muscle stimulation techniques and quantitative electromyography.

Recovery from neuromuscular disorder

Some authors advise against adoption of electrophysiological differential diagnosis [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Hermans | Impact of intensive insulin therapy on neuromuscular complications and ventilator dependency in the medical intensive care unit[END_REF] since distinguishing between CIM and CIP would not be associated with clinical prognosis. We recently reported that CIM constitutes the primary reason for ICU acquired weakness presenting in critically ill patients suffering from sepsis, systemic inflammatory response syndrome or multiple organ failure once sedation is ended. [START_REF] Weber-Carstens | Non-excitable muscle membrane predicts ICUacquired paresis in mechanically ventilated, sedated patients[END_REF] For the first time we report that ICU length of stay is markedly prolonged in patients classified as CIM/CIP compared to patients with isolated CIM and that this does not result from illness severity after sedation was ended. Expecting subsequent prolongation of recovery time, it was interesting to observe that patients classified as CIM/CIP still featured severe weakness at ICU discharge in contrast to CIM patients. This is consistent with observations by Guarneri and colleagues describing better long-term prognosis (one year after hospital discharge) in patients diagnosed with isolated CIM. [START_REF] Guarneri | Long-term outcome in patients with critical illness myopathy or neuropathy: The Italian multicentre CRIMYNE study[END_REF] It should nevertheless be mentioned that patients classified as CIM/CIP showed more pronounced dmCMAP amplitude reduction and shorter MUAP duration, both characteristics of pronounced myopathy, than patients with pure CIM.

As some CIM patients show recovery of dmCMAP amplitude reduction at ICU discharge, we presume inactivation of voltage-gated sodium channels in structural intact muscle fibres to be reversible after successful treatment/elimination of potentially involved factors, which would explain return to normal function within days. [START_REF] Bednarik | Critical illness polyneuromyopathy: the electrophysiological components of a complex entity[END_REF][START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF][START_REF] Rich | Direct muscle stimulation in acute quadriplegic myopathy[END_REF]38] Recovery was not observed in patients classified as CIM/CIP, which may be either due to pronounced muscle dysfunction with selective myosin-filament loss in fast twitch muscle fibres OR due to muscle denervation causing muscle membrane depolarization potentially counteracting recovery. [START_REF] Trojaborg | Electrophysiologic studies in critical illness associated weakness: myopathy or neuropathy -a reappraisal[END_REF]36,38,[START_REF] Guth | The roles of disuse and loss of neurotrophic function in denervation atrophy of skeletal muscle[END_REF] In conclusion, we were able to show that clinical prognosis differs according to electrophysiological differential diagnosis during early critical illness. CIM in combination for ICU-control, CIM-and CIM/CIP-patients. Differences are shown for CIM/CIP patients vs. purely CIM-patients (Kruskal-Wallis test). neCMAP = nerve evoked compound action potential amplitude, dmCMAP = direct muscle stimulated compound action potential amplitude.


Prof. Dr Christoph Helmchen
email: christoph.helmchen@neuro.uni-luebeck.de

Jan C Klinkenstein

Alexandra Krüger

Jan Gliemroth

Christian Mohr

Thurid Sander

M.D Neuroimage

Structural brain changes following

Keywords: vestibular lesion, vestibular compensation, voxel-based morphometry

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Central processing of vestibular function is crucially determined by bilateral peripheral input. Vestibular tone imbalance reflects a pathological asymmetry of bilateral vestibular activity in the brain. In the acute stage vestibular tone imbalance causes severe direction-specific oculomotor (spontaneous nystagmus), perceptional (vertigo) and postural deficits (ipsilesional falls). It remains a matter of debate why patients with the same persistent peripheral vestibular deficit show quite different vestibular recovery. The peripheral vestibular deficit alone does not seem to determine functional recovery. [START_REF] Curthoys | Vestibular compensation and substitution[END_REF] Despite only partial or incomplete restoration of neural activity in the peripheral vestibular afferents many patients with vestibular neuritis or unilateral surgical vestibular lesions recover probably due to mechanisms of central compensation. [START_REF] Curthoys | Vestibular compensation and substitution[END_REF][START_REF] Dieterich | Functional brain imaging of peripheral and central vestibular disorders[END_REF] Vestibular rehabilitation may not only involve restoration of peripheral vestibular function but also compensation by central recalibration [START_REF] Palla | Recovery of the high-acceleration vestibulo-ocular reflex after vestibular neuritis[END_REF][START_REF] Schmid-Priscoveanu | Caloric and searchcoil head-impulse testing in patients after vestibular neuritis[END_REF] , or substitution, i.e. by the somatosensory 5 or visual system. [START_REF] Dieterich | Evidence for cortical visual substitution of chronic bilateral vestibular failure (an fMRI study)[END_REF] While there is some physiological, clinical [START_REF] Curthoys | Vestibular compensation and substitution[END_REF][START_REF] Palla | Recovery of the high-acceleration vestibulo-ocular reflex after vestibular neuritis[END_REF] and functional imaging [START_REF] Bense | Metabolic changes in vestibular and visual cortices in acute vestibular neuritis[END_REF] evidence for vestibular compensation following unilateral peripheral vestibular lesions [START_REF] Dieterich | Functional brain imaging of peripheral and central vestibular disorders[END_REF] , little is known about structural cerebral changes that might also account for the great variability in vestibular recovery. First evidence for structural brain changes following chronic vestibular deafferentation was found in patients with bilateral vestibulopathy. [START_REF] Brandt | Vestibular loss causes hippocampal atrophy and impaired spatial memory in humans[END_REF] Based on a very specific a priori hypothesis of spatial memory and navigation deficits in patients with peripheral vestibular failure, patients with bilateral [START_REF] Brandt | Vestibular loss causes hippocampal atrophy and impaired spatial memory in humans[END_REF] but not unilateral [START_REF] Hufner | Spatial memory and hippocampal volume in humans with unilateral vestibular deafferentation[END_REF] vestibulopathy showed hippocampal atrophy. In a recent study, we could identify grey matter changes in multisensory vestibular cortex areas of patients with vestibular neuritis which correlated with the clinical vestibular function and vertigorelated disability. [START_REF] Helmchen | Structural changes in the human brain following vestibular neuritis indicate central vestibular compensation[END_REF] Therefore the aim of this study in patients with a unilateral, irreversible peripheral vestibulo-cochlear lesion was to identify structural brain changes which may be related to auditory deficits and / or functional vestibular impairment and disability. We examined patients after unilateral vestibular lesion due to acoustic neuroma (AN) and hypothesized that putative structural brain changes may be related to functional disability possibly indicating vestibular and auditory compensation. We focussed our analysis on cortical structures involved in vestibular and auditory processing: Heschl gyrus, the superior temporal gyrus (STG), the inferior parietal lobule (IPL), posterior insula, anterior cingulate cortex, primary (S1) and secondary somatosensory cortex (S2), middle temporal area (MT/V5) and hippocampus, and cerebellum, most of which belong to a multisensory vestibular cortical network in man. [START_REF] Dieterich | Functional brain imaging: a window into the visuo-vestibular systems[END_REF] They correspond closely to temporo-insular and temporo-parietal multisensory areas in the monkey [START_REF] Guldin | Is there a vestibular cortex?[END_REF] responding to vestibular, visual and somatosensory stimuli. These areas have been identified in previous activation studies using PET or fMRI following galvanic [START_REF] Stephan | Functional MRI of galvanic vestibular stimulation with alternating currents at different frequencies[END_REF][START_REF] Bense | Multisensory cortical signal increases and decreases during vestibular galvanic stimulation (fMRI)[END_REF] , caloric [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF][START_REF] Fasold | Human vestibular cortex as identified with caloric stimulation in functional magnetic resonance imaging[END_REF] or click-evoked [START_REF] Schlindwein | Cortical representation of saccular vestibular stimulation: VEMPs in fMRI[END_REF] vestibular stimulation in healthy subjects as well as in patients with peripheral [START_REF] Bense | Preserved visual-vestibular interaction in patients with bilateral vestibular failure[END_REF] and central vestibular lesions [START_REF] Dieterich | Medial vestibular nucleus lesions in Wallenberg's syndrome cause decreased activity of the contralateral vestibular cortex[END_REF][START_REF] Dieterich | Thalamic infarctions cause side-specific suppression of vestibular cortex activations[END_REF] .

METHODS

We compared structural magnetic resonance images (T1-weighted three-dimensional gradient echos acquired with a fast low angle shot sequence [FLASH]) of patients following unilateral acoustic neuroma (AN) removal with individually age-and gendermatched healthy control subjects. To relate relative GMV changes of VBM to the clinical disability we used scores of objective and subjective vestibular impairment (see below).

Subjects

We studied 15 right-handed patients (4 male, 11 female) who suffered from a unilateral (left n=10; right n=5) acoustic neuroma (AN). Apart from AN, patients had no history of neurological or neuro-otological disease. All of them had a long-standing history (6.25 +/-3.9 years) of hearing impairment, tinnitus or dizziness (Table 1).

Tumors were surgically removed in all of them leaving most of the patients with severe unilateral peripheral vestibular lesions postoperatively. Patients were examined on average 37 ± 26 months (range: 3-81 months) after the operation using a battery of clinical neurological and neuro-otological tests, electrophysiological investigations, clinical disability scores and VBM (T1 GE 3D FLASH). Most patients had pronounced hypacusis or anacusis on the operated side following surgery (Table 1). We did not include patients with absent peak I wave in the auditory evoked potentials. Patients showed a variable degree of facial palsy on the operated side (Table 1). Functional parameters (i.e. caloric paresis, vestibular disability score) were correlated with changes in grey matter volume. Fifteen age-and gender-matched healthy subjects (4 male, 11 female, all right-handed) served as pair wise controls (individual matching between patient and control with ± 2 years maximal difference).

Healthy control subjects were recorded in the same interval and with the same MRI protocol as the patients. They had no history of neurological or vestibulo-cochlear disease and were normal on neuro-otological clinical examination. The age range of both groups was 35 -67 years (mean age of AN patients: 56.6 ± 8.9 years; healthy control subjects 55.7 ± 9.1 years). Except for the antivertiginous medication (e.g. dimenhydrinate) in the first four days after surgery, patients did not receive any additional medication. In the hospital, patients had physiotherapy (modified version of Tjernstrom [START_REF] Tjernstrom | Vestibular PREHAB and gentamicin before schwannoma surgery may improve long-term postural function[END_REF] ) but they did not have standardized professional physiotherapy after their discharge from the hospital. All subjects gave written informed consent for participation in the study which was conducted in accordance with the declaration of Helsinki and approved by the Ethics committee of the University of Luebeck.

Clinical examinations

All subjects underwent a detailed neurological and neuro-otological examination by two independent, clinically experienced specialists of the University's vertigo unit.

Except for partial ipsilesional facial palsy none of the patients had neurological signs beyond those resulting from the AN (hypacusis, signs of peripheral vestibulopathy), particularly there were no clinical signs of brainstem disease.

Neuro-otological signs of vestibular imbalance were assessed in a standardized fashion using a clinical vestibular score (CVS) [START_REF] Helmchen | Structural changes in the human brain following vestibular neuritis indicate central vestibular compensation[END_REF] . This score is based on clinical examination covering (1) spontaneous nystagmus, (2) head-shaking nystagmus, (3) gaze-evoked nystagmus, (4) positional nystagmus, (5) head impulse test [START_REF] Halmagyi | A clinical sign of canal paresis[END_REF] , (6) ataxia of stance, (7) lateropulsion (8) lateropulsion during gait without horizontal head movements and (9) ataxia of gait with horizontal head movements, (10) tandem gait ataxia, (11) ataxia on body rotation (180 deg), and (12) the Fukuda stepping test.

Each subtest was scored with "0" for absent, "1" for moderate and "2" for severe manifestation of the clinical sign. Accordingly, CVS could range from zero (normal) to 24 (maximum). Values above 3 were taken as pathological (range in healthy control subjects: 0-3).

Self-assessment of vestibular disability was performed using the Cohen and Kimball score (using 28 items consisting of instrumental, ambulation and functional tasks which were evaluated on a 0-10 scale with 0=normal). [START_REF] Cohen | Development of the vestibular disorders activities of daily living scale[END_REF]

Recordings of vestibular and hearing function

In addition to the head impulse test (Halmagyi-Curthoys test) [START_REF] Halmagyi | A clinical sign of canal paresis[END_REF] peripheral vestibular excitability was tested by bithermal caloric irrigation and horizontal (yaw) rotation in the dark using DC electro-oculography. As a measure of unilateral vestibular loss, the mean peak slow-phase velocity during caloric irrigation with water at 30°C and 44°C was measured. Caloric hyporesponsiveness was calculated with the Jongkees's vestibular paresis formula. [START_REF] Jongkees | Clinical nystagmography: a detailed study of electro-nystagmography in 341 patients with vertigo[END_REF] Only patients with at least a 25 % canal paresis were included. Combined semicircular canal and otolithic function in the frontal (roll) plane was assessed by the subjective visual vertical (SVV) as described elsewhere. [START_REF] Rambold | Differential vestibular dysfunction in sudden unilateral hearing loss[END_REF] Hearing function was assessed by pure tone audiogram and auditory evoked potentials. [START_REF] Rambold | Differential vestibular dysfunction in sudden unilateral hearing loss[END_REF] Hearing impairment at the time of VBM recordings was classified according to the hearing score by Gardner and Robertson 26 , with "5" indicating deafness, "4" refering to >90 dB threshold in pure tone audiometry, "3" covering 51-90 dB, "2" 31-50 dB, and "1" with 0-30 dB reflecting normal hearing.

MRI scanning and preprocessing

All high-resolution anatomical 3D images were acquired, preprocessed and analysed as described elsewhere [START_REF] Helmchen | Structural changes in the human brain following vestibular neuritis indicate central vestibular compensation[END_REF] . T1-weighted three-dimensional gradient echos acquired with a fast low angle shot sequence [FLASH], TR = 15 ms, TE = 5 ms, flip angle 30°, acquisition matrix 256 x 256, isotropic voxel = 1 mm 3 ) were acquired on a 1.5 T scanner (Siemens Symphony, Erlangen, Germany) with a standard head coil to reduce possible inhomogeneities and geometric variations due to magnetic field and gradient differences. Images were then analyzed using the optimized VBM protocol [START_REF] Good | Cerebral asymmetry and the effects of sex and handedness on brain structure: a voxel-based morphometric analysis of 465 normal adult human brains[END_REF] with the VBM toolbox for SPM2 (http://www.fil.ion.ucl.ac.uk/spm, Welcome Dept. of Imaging Neuroscience, London, UK), a fully automated technique for computational analysis of relative differences in local grey matter volume. Preprocessing of data involved coregistration, customized study template creation, spatial normalization, segmentation and spatial smoothing. [START_REF] Ashburner | Voxel-based morphometry--the methods[END_REF] First, anatomical images of patients with acoustic neurinoma (AN) on the right side were mirrored on the left side to guarantee a homogenous group. Accordingly, images of corresponding (age-/ gender-matched) control subjects for these patients were also mirrored. All data are calculated for leftsided AN, with left-sided GMV changes being ipsilesional due to surgically acquired deficits of vestibular and cochlear function. Images were coregistered to the standard MNI template (Montreal Neurolocical Institute, Montreal, Canada) of SPM2. All anatomical images were then used to create a customized grey matter anatomical study template to determine the anatomy and reduce scanner-and populationspecific bias. This template was smoothed with a Gaussian kernel of 8 mm full-widtha-half-maximum (FWHM). Spatial normalization of all images to this customized study template was performed using a nonlinear spatial transformation to remove differences in overall size, position, global space and to transfer images to the MNI space, which approximates the Talairach space. This normalization improved the overlap of corresponding anatomical structures. [START_REF] Hellier | Retrospective evaluation of intersubject brain registration[END_REF] Finally, using a modified mixture model cluster analysis, normalized images were corrected for non-uniformities in signal intensity and partitioned in grey (GM), white matter, cerebrospinal fluid (CSF) and background. To remove unconnected non-brain voxels, we applied a series of morphological erosions and dilatations to the segmented images. [START_REF] Good | Cerebral asymmetry and the effects of sex and handedness on brain structure: a voxel-based morphometric analysis of 465 normal adult human brains[END_REF][START_REF] Good | A voxel-based morphometric study of ageing in 465 normal adult human brains[END_REF] No additional modulations were applied. The resulting grey images were smoothed with a Gaussian kernel of 12 mm FWHM. To account for age-related GMV changes, age of all subjects was introduced as a covariate of interest in the analysis.

Statistical analysis and correlation

Data of grey matter was analyzed using voxel-by-voxel multiple regression model (AnCova, VBM with SPM2) in a cross-sectional data analysis revealing differences in relative local GMV changes across the whole brain between AN patients and controls. To account for age-related GMV changes, age of all subjects was introduced as a covariate of interest in the analysis. We excluded all voxels of GMV of < 0.2 (of a maximum value of 1) to obtain fairly homogenous voxels. Because of our strong a priori hypothesis we focused our analysis of regional relative grey matter volume (GMV) changes in the regions of interest (ROI): thalamus, hippocampus, primary (S1) and secondary somatosensory cortex (S2), cerebellum, auditory cortex (Heschl gyrus), posterior insula, inferior parietal lobe (IPL), superior temporal gyrus, visual cortex, middle temporal area (MT/V5) most of which belong to a multisensory vestibular cortical network in man [START_REF] Dieterich | Functional brain imaging: a window into the visuo-vestibular systems[END_REF] . They correspond closely to temporo-insular and temporo-parietal multisensory areas in the monkey responding to vestibular, visual and somatosensory stimuli. They have been identified in previous activation studies using PET or fMRI following galvanic [START_REF] Stephan | Functional MRI of galvanic vestibular stimulation with alternating currents at different frequencies[END_REF] , caloric [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF][START_REF] Fasold | Human vestibular cortex as identified with caloric stimulation in functional magnetic resonance imaging[END_REF] or click-evoked [START_REF] Schlindwein | Cortical representation of saccular vestibular stimulation: VEMPs in fMRI[END_REF] vestibular stimulation in healthy human subjects as well as in patients with peripheral [START_REF] Bense | Preserved visual-vestibular interaction in patients with bilateral vestibular failure[END_REF] and central vestibular lesions [START_REF] Dieterich | Medial vestibular nucleus lesions in Wallenberg's syndrome cause decreased activity of the contralateral vestibular cortex[END_REF][START_REF] Dieterich | Thalamic infarctions cause side-specific suppression of vestibular cortex activations[END_REF] . Regions of interest (ROI) were created by using the ROI-tool of the WFU-Pick-Atlas in SPM. Regions were defined by appropriate spheres (r between 15 and 30 mm) centered in the middle of the corresponding brain region. Twenty-three ROIs were used including 21.6 % (47585 / 221379 voxel) of the total brain volume. We generated a mask including those regions to identify relative GMV alterations between AN patients and controls according to our hypothesis. This mask was applied to the multiple regression model (AnCova) in a second step for a priori ROI based statistical analysis for significant relative GMV differences between AN patients and controls. Absolute GMV changes were not calculated.

Statistics inside this ROI analysis were considered significant with p < 0.005 (uncorrected) and spatial extent > 200 voxel (ROI-analysis). Reported changes in grey matter volume did not survive a more conservative statistical threshold using corrections for multiple comparisons (FWE or FDR correction) best explained by the small study population (n = 15). High voxel threshold was determined to reduce false positive minor clusters [START_REF] Worsley | Detecting changes in nonisotropic images[END_REF] . In a third step we performed correlations (simple regression, SPM2) among the grey matter volume data (ROI) of the AN patients with their scores of vestibular and cochlear function (Table 2). Values of scores were mean corrected before entering the correlation analyses. These correlations were analyzed to elucidate the functional significance of the GMV changes. Correlations are considered significant for p < 0.005 unless otherwise stated. Thus, due to the limited population size, GMV data were used uncorrected for multiple comparisons, but their significance is underlined by the correlations. The Wake Forest University (WFU)-Pick Atlas [START_REF] Maldjian | An automated method for neuroanatomic and cytoarchitectonic atlas-based interrogation of fMRI data sets[END_REF] was used to determine anatomical structures of GMV changes.

RESULTS

Patients

Clinical data of patients following surgical removal of AN are listed in Table 1. All patients had clinical signs of unilateral peripheral vestibular deficit, i.e. all patients showed a pathological head impulse test to the side of the operation. All patients showed severe caloric hyporesponsiveness [91.2 ± 16.27 %; 100 %=no caloric response) and severe hearing impairment (on average 4 ± 1.5 using the tonaudiometry of the Gardner-Score) on the lesion side (operation). Contralesional auditory and vestibular function was normal (Halmagyi-Curthoys test, caloric irrigation). Auditory evoked potentials yielded a normal peak I (amplitude and latency) and delayed or absent peaks II-V on the side of neurectomy while the contralesional side was normal. Subjective visual vertical (SVV) was on average within normal limits (< 2°) [START_REF] Rambold | Differential vestibular dysfunction in sudden unilateral hearing loss[END_REF] . The clinical vestibular impairment score (CVS) was on average 8.0 ± 4.9 (0=normal, 24=complete vestibular deficit). Vestibular disability [START_REF] Cohen | Development of the vestibular disorders activities of daily living scale[END_REF] was scored on average 3.0 +/-1.5. Clinical scores of vestibular deficits and disability (CVS and Cohen Score) did not correlate with caloric hyporesponsiveness. CVS was neither correlated with the duration of symptoms nor the interval (months) since neurectomy.

Imaging data

The categorical comparisons revealed relative GMV-changes in brain regions of patients as compared to age-matched control subjects (Table 2). Apart from the inferior parietal and inferior and middle temporal lobe, there were bilateral GMV increases in bilateral primary somatosensory cortex (S1) and visual motion processing areas, i.e. middle temporal area (MT / V5) (Fig. 1) and the cerebellar vermis (lobule VII). Relative decreases were found in frontal, temporal lobe and in the cerebellum (Table 2). There were no significant changes in hippocampus but the absence of a statistically significant effect in a particular region does not prove that the region is unaffected [START_REF] Ridgway | Ten simple rules for reporting voxel-based morphometry studies[END_REF] . No age-related effects were found (last two bars on the right in Fig. 1 indicate age as a covariate of interest).

Clinical correlations with the clinical assessment (CVS, hearing impairment) or the vestibular disability score were calculated using linear regression analyses.

Significant correlations with clinical scores were only found for relative GMV increases but not for decreases. Details of the morphometric analysis are given in Table 2.

There was a contralesional GMV increase in the superior temporal gyrus (STG) and adjacent posterior insula which correlated negatively with CVS (Fig. 2A): patients with only subtle signs of vestibular impairment showed the largest relative increase in GMV. Conversely, relative GMV increase was smallest or absent with the largest scores of vestibular impairment resulting from the peripheral vestibular lesions. Functional disability [START_REF] Cohen | Development of the vestibular disorders activities of daily living scale[END_REF] was negatively correlated with relative GMV increase in the inferior parietal lobe (IPL) (Fig. 2B), i.e., GMV increase became smaller with increasing functional disability or, conversely, relative GMV increase was strongest with the least disability.

Relative GMV increases correlating with hearing impairment (Gardner Score) were found in the contralesional auditory cortex (Heschl gyrus) and adjacent posterior insula (Fig. 2C). Relative GMV increase was largest with the least hearing impairment and there was no or little GMV increase with hearing loss. This increase was found on the contralesional side but there was a trend towards bilateral increase which just failed significance on the ipsilesional side. Correlations between relative GMV changes and the SVV were not performed as SVV was within normal limits.

With respect to the major effects of relative GMV increases there were no correlations of GMV changes in MT/V5 or somatosensory cortex with clinical or subjective parameters. There were no correlations of GMV changes with the Brackmann Score of facial palsy, the size of the resected tumor or the duration lapsed since the operation.

DISCUSSION

Unilateral vestibulo-cochlear lesion elicited an increase of GMV in visual motionsensitive areas (MT/V5) and in the primary somatosensory cortex as well as additional increases of GMV in regions of the multisensory vestibular cortex areas which correlated with clinical and electrophysiological parameters of vestibulocochlear impairment. This provides some first evidence that peripheral vestibular lesions elicit structural brain changes which might be related to substitution and compensation.

Acute vs. chronic vestibular lesions

Unilateral peripheral vestibular lesions elicit a vestibular tone imbalance not only at the level of the vestibular nuclei but also at the level of the thalamus [START_REF] Dieterich | fMRI signal increases and decreases in cortical areas during small-field optokinetic stimulation and central fixation[END_REF] and the "primary" (e.g. posterior insula) and "secondary" (e.g. STG, IPL) vestibular cortex 2 . In the acute stage this tone imbalance can be detected at cortical levels: using positron emission tomography (PET), patients with vestibular neuritis (VN) showed increased regional cerebral glucose metabolism (rCGM) in the contralesional multisensory vestibular cortex. [START_REF] Bense | Metabolic changes in vestibular and visual cortices in acute vestibular neuritis[END_REF] This activation resembles vestibular stimulation in healthy subjects [START_REF] Dieterich | Functional brain imaging of peripheral and central vestibular disorders[END_REF] which is stronger in the hemisphere ipsilateral to vestibular stimulation. [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF] This reflects an inherent physiological dominance of ipsilateral vestibular projections in bilateral cortical representation. [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF] Accordingly, ipsilateral vestibular projection is deficient in acute vestibular neuritis giving rise to a vestibular tone imbalance with contralesional rCGM increase due to persisting contralesional vestibular resting discharge.

Clinical signs of vestibular imbalance improve gradually within the first two months probably due to central compensation 2 and, accordingly, rCGM normalizes after 3 months in vestibular neuritis. [START_REF] Bense | Metabolic changes in vestibular and visual cortices in acute vestibular neuritis[END_REF] At this time, 3 months after vestibular neuritis, morphological changes of GMV can be identified in the multisensory vestibular cortex of VN patients. [START_REF] Helmchen | Structural changes in the human brain following vestibular neuritis indicate central vestibular compensation[END_REF] The contralesional increase in GMV in retroinsular and posterior insula cortex and STG of VN patients might be the consequence of continuous contralesional increase of rCGM during the acute phase of vestibular neuritis. [START_REF] Bense | Metabolic changes in vestibular and visual cortices in acute vestibular neuritis[END_REF] The asymmetry of neural activity is probably a driving force for vestibular compensation.

Notably, this may be functionally significant as VN patients with large GMV increases showed least vestibular impairment. As peripheral regeneration (caloric responsiveness) is a rather poor indicator for vestibular compensation [START_REF] Palla | Vestibular neuritis: Vertigo and the highacceleration vestibulo-ocular reflex[END_REF] GMV changes may be more valuable signs and possibly even contribute to functional recovery.

Here, we demonstrate that AN patients with unilateral peripheral vestibular lesion showed two changes contributing to the morphological substrate of a vestibular imbalance at cortical levels. First, GMV decreases were found in parts of the ipsilesional "secondary" vestibular cortex, e.g. STG and fusiform gyrus. This is also in accord with a deficient dominance of ipsilateral vestibular projections to the cortex. [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF] Second, there was a contralesional increase of GMV in the posterior insula. This is in accord with the failure of the ipsilateral dominance of vestibular projections 15 using PET [START_REF] Bense | Metabolic changes in vestibular and visual cortices in acute vestibular neuritis[END_REF] and VBM recordings 10 : the contralesional increase in rCGM is associated with an increase in GMV which may contribute to vestibular compensation as the increase was larger the better patients recovered from vestibular tone imbalance. As the vestibular system has a dominant representation in the non-dominant hemisphere [START_REF] Dieterich | Dominance for vestibular cortical function in the non-dominant hemisphere[END_REF] one might suspect this to be the reason for the right-hemispheric GMV changes being correlated with clinical impairment (scores) in our patients. However, as GMV changes became stronger the less patients were clinically affected, we suspect compensation rather than hemispheric dominance as the responsible mechanism.

Unlike VN patients, our AN patients had substantial reduction of peripheral vestibular function which may be related to the surgical tumor resection or the preceding tumor growth. This probably accounts for the lack of GMV changes being correlated with caloric hyporesponsiveness. Additional evidence for vestibular compensation comes from structural changes in the inferior parietal lobe (IPL, BA40) which contains vestibular related neural activity [START_REF] Schlindwein | Cortical representation of saccular vestibular stimulation: VEMPs in fMRI[END_REF][START_REF] Bremmer | Space coding in primate posterior parietal cortex[END_REF] and showed bilateral, predominantly contralesional increase in GMV; the larger the increase the less patients were functionally disabled.

Auditory lesions

Ascending projections of the auditory system to the cortex are generally thought to be bilateral. Using fMRI [START_REF] Schlindwein | Cortical representation of saccular vestibular stimulation: VEMPs in fMRI[END_REF][START_REF] Overath | Encoding of spectral correlation over time in auditory cortex[END_REF] , unilateral acoustic stimulation elicited bilateral but preferentially contralateral [START_REF] Schlindwein | Cortical representation of saccular vestibular stimulation: VEMPs in fMRI[END_REF] activation of the primary auditory cortex, i.e., the transverse temporal gyrus of Heschl in the depth of the Sylvian fissure. Little is known about the cortical and subcortical auditory tone imbalance following AN surgery. Here we demonstrate GMV increase in the contralesional auditory cortex (medial aspects of the Heschl gyrus) which was larger the less patients had hearing impairments. At present we cannot conclude whether this is related to an improvement of central hearing processing (e.g. in the attempt to improve signal detection [START_REF] Strelnikov | Does Brain Activity at Rest Reflect Adaptive Strategies? Evidence from Speech Processing after Cochlear Implantation[END_REF] ) or reflects the morphological response to an innervation tone imbalance in the auditory system elicited by the unilateral partial deafferentation.

Changes in non-vestibular sensory systems

Somatosensory substitution

GMV increases in motion-sensitive areas (MT/V5) and primary somatosensory cortices may reflect upregulation of sensory interactions since patients with irreversible unilateral peripheral vestibulo-cochlear lesions rely stronger on other sensory afferents. [START_REF] Dieterich | Functional brain imaging of peripheral and central vestibular disorders[END_REF] Upregulation of the sensitivity of other sensory systems, particularly the somatosensory system has been described before [START_REF] Parietti-Winkler | Sensorimotor postural rearrangement after unilateral vestibular deafferentation in patients with acoustic neuroma[END_REF] . There is a gradual buildup over weeks of an asymmetrical (ipsilesional) increase in muscle spindle input following unilateral vestibular lesion which demonstrates a unilateral increase in somatosensory weight, which may substitute for the missing vestibular input. [START_REF] Strupp | Perceptual and oculomotor effects of neck muscle vibration in vestibular neuritis. Ipsilateral somatosensory substitution of vestibular function[END_REF] Our AN patients showed bilateral increase of GMV in the primary somatosensory cortex (S1) which might reflect an increase in the somatosensory weight following chronic peripheral vestibular deafferentation as patients have to rely more on somatosensory cues to stabilize balance, i.e. a substitution of the vestibular deficit by other non-vestibular systems.

Visual-vestibular interaction

Reciprocal inhibition between different sensory systems has been proposed as a fundamental working principle of the central nervous system in health and disease [START_REF] Dieterich | Functional brain imaging of peripheral and central vestibular disorders[END_REF] and there is accumulating evidence for multisensory, e.g. visual-auditory, somatosensory-visual and visual-vestibular reciprocal interaction. For example, suppression of visual cortex activity may be important to reduce oscillopsia caused by vestibular nystagmus resulting from acute vestibular tone imbalance. [START_REF] Brandt | Reciprocal inhibitory visualvestibular interaction. Visual motion stimulation deactivates the parieto-insular vestibular cortex[END_REF] We found a bilateral increase of GMV in middle temporal visual area (MT/V5). MT/V5 is involved in reciprocal visual-vestibular interaction. [START_REF] Dieterich | fMRI signal increases and decreases in cortical areas during small-field optokinetic stimulation and central fixation[END_REF][START_REF] Brandt | Reciprocal inhibitory visualvestibular interaction. Visual motion stimulation deactivates the parieto-insular vestibular cortex[END_REF] Unlike MST and somatosensory cortex, MT does not contain vestibular neurons (Dora Angelaki, personal communication). Accordingly, the increase in GMV in MT may be related to non-vestibular substitution of the peripheral vestibular deficit. It might be a morphological consequence of the altered visual sensitivity and visual motion perception in patients with peripheral vestibular failure. [START_REF] Dieterich | Evidence for cortical visual substitution of chronic bilateral vestibular failure (an fMRI study)[END_REF][START_REF] Deutschlander | Unilateral vestibular failure suppresses cortical visual motion processing[END_REF] Limitations of the study It was the aim of this first study to investigate putative GMV changes which correlate with parameters of vestibular impairment. We are aware that specificity of compensation is additionally provided by longitudinal studies covering pre-and postoperative investigations at different intervals. Moreover, vestibular afferents were partially preserved in the tumor operation (incomplete loss of caloric excitability) which could have contributed to compensation, in particular if one takes the fairly large range of intervals between surgery and fMRI recordings into account. However, there was neither a correlation of GMV with caloric responsiveness nor with the duration since the operation. Finally, for ethical reasons, all patients received physiotherapy in the acute postoperative stage but based on our results future studies should investigate the effect of (different forms of) physiotherapy on these GMV changes. Importantly, all variables listed above could have been confounds if we had not found any correlation. However, as we did, our interesting correlations now deserve further investigations.

Figures

Table 2:

Significant relative grey matter (I) changes are shown for the left (ipsilesional, il) and the right (contralesional, cl) hemisphere. The x ,y ,z coordinates are given in standard Montreal Neurological Institute (MNI) space and the anatomical regions according to the Wake Forest University (WFU) Pick Atlas. Each region is defined by the local maximum within a cluster. Simple regression analyses revealed significant correlations of grey matter volume (GMV) changes in patients (II) with (a) clinical vestibular score (CVS), (b) vestibular disability (Cohen and Kimball Score), and (c) the hearing loss (Gardner Score).

Fig. 1 :

 1 Fig.1: Regions of significant changes in relative grey matter volume (GMV) (patients vs. controls) are shown on representative axial, sagittal or coronal slices of the standard Montreal Neurological Institute (MNI) template. The color-bar represents the T statistics for plotted results (A to D). A relative increase in GMV is indicated by the hot-iron color map. The coordinates of the local maxima for each cluster of significant relative GMV-change revealed by categorical comparison of patients vs. controls are given on the ordinate. The barplots (right) illustrate the relative change in GMV of patients compared to controls in arbitrary units ± standard error of mean (SEM) for patients (first bar), controls (second bar) and corresponding correlation with the age of patients (third bar) and controls (fourth bar) indicating that age does not account for the GMV changes. GMV increase is shown in the ipsi-(A) and contralesional (B) primary somatosensory cortex (S1), (C) bilaterally in motion sensitive areas (MT/V5) and (D) in lobule VII of the cerebellar vermis. Left side in slices is indicated by "L" (ipsilesional), "R" indicates right side (contralesional).

Fig. 2 :

 2 Fig.2: Significant correlations of grey matter volume (GMV) increase in the superior temporal gyrus (STG) / posterior insula (A) with the clinical vestibular score (CVS), in the inferior parietal lobule (IPL) (B) with the vestibular disability (Cohen and Kimball score), and in the contralesional Heschl gyrus / posterior insula (C) with the hearing loss (Gardner score) are shown on representative axial and coronal slices of the standard Montreal Neurological Institute (MNI) template and indicated by arrows (x-, z-coordinates below the slices). T-scores of the correlation are indicated by the hotiron color map. On the right side, the linear regressions are plotted. Left side in slices is indicated by "L" (ipsilesional), "R" indicates right side (contralesional). R 2 = coefficient of determination.

Acknowledgements

TS was supported by a research grant E04-2009 from the University of Luebeck.

Table 1:

Clinical data of patients with unilateral vestibular-cochlear failure following operative removal of acoustic neuroma. F= female; M = male, L = left; R = right; VBM = voxel based morphometry; for scores see Methods (caloric hyporesponsiveness: 0-100%; 100%= no caloric response; vestibular disability score: range 0-10, 10 = most severely affected; CVS=Clinical score of vestibular signs: range 0-24, 24 = most severely affected; House Brackmann Score of facial palsy: range 0-6). Head impulse test (