Béla Büki
email: bukibela@hotmail.com

Sándor Garab

Yunxia W Lundberg

Keywords: subjective, paroxysmal, positioning, vertigo, otolith Word count: 3482

Presently, the unambiguous diagnosis of benign paroxysmal positioning vertigo (BPPV) requires the detection of positioning or positional nystagmus provoked by Dix-Hallpike (for vertical semicircular canals) or supine roll (for horizontal semicircular canals) maneuvers, which indicates canalo-or cupolithiasis of affected semicircular canals. There are patients, however, in whom -despite typical complaints of BPPV -no positional nystagmus can be documented; this is called "subjective BPPV" (sBPPV). These patients usually complain of short vertigo spells during and after sitting up, sometimes with abnormal retropulsion of the trunk. In this study, we aimed to ascertain whether these patients in fact demonstrate abnormal sitting-up trunk oscillations when measured by posturography. Of 200 unselected patients with vertigo or dizziness, 43% had sBPPV with vertigo spells during sitting up, and 20% classical BPPV. Posturographic recordings were performed in 20 patients with sBPPV and sitting-up vertigo. Among them 7 had trunk oscillations during the act of sitting up and for a short time immediately afterwards. Based on our findings, we propose a new type of BPPV, the so-called Type 2 BPPV (typical complaints of BPPV, no nystagmus in Dix-Hallpike positions but short vertigo spell during sitting up), which may be the result of chronic canalolithiasis within the short arm of a posterior canal. Furthermore, we suggest that Type 2 BPPV, which could be identical to sBPPV or constitute a major subgroup of it, occurs frequently among patients with vertigo. For therapy we recommend repetitive sit-ups from the Dix-Hallpike positions to liberate the short arm of the posterior canal from canaloliths.

INTRODUCTION

Benign paroxysmal positional vertigo (BPPV) is the most common neuro-otological disorder. [START_REF] Mizukoshi | Epidemiological studies on benign paroxysmal positional vertigo in Japan[END_REF][START_REF] Froehling | Benign positional vertigo: incidence and prognosis in a population-based study in Olmsted County, Minnesota[END_REF][START_REF] Oghalai | Unrecognized benign paroxysmal positional vertigo in elderly patients[END_REF][START_REF] Von Brevern | Epidemiology of benign paroxysmal positional vertigo: a population based study[END_REF][START_REF] Katsarkas | Benign paroxysmal positional vertigo (BPPV): idiopathic versus posttraumatic[END_REF][START_REF] Mosca | Benign paroxysmal positional vertigo, incidence and treatment[END_REF] Presently, the definition of BPPV requires vertical-torsional positional nystagmus evoked by the Dix-Hallpike maneuver or predominantly horizontal positional nystagmus after rolling the head sideways from the supine position. [START_REF] Bhattacharyya | Clinical practice guideline: benign paroxysmal positional vertigo[END_REF][START_REF] Parnes | Diagnosis and management of benign paroxysmal positional vertigo (BPPV)[END_REF][START_REF] Aw | Benign positional nystagmus: a study of its three-dimensional spatio-temporal characteristics[END_REF] The former nystagmus indicates canalo-or cupulolithiasis of posterior or rarely anterior semicircular canals, while the latter nystagmus is seen in canalo-or cupulolithiasis of the horizontal canals.

Neuro-otologists regularly see patients with typical complaints of BPPV, but without positioning nystagmus, even with repeated provocatory maneuvers. Cases with BPPV symptoms without positioning nystagmus have been termed "subjective BPPV" (sBPPV). The fact that repositioning maneuvers are effective in patients with sBPPV supports the notion that sBPPV, like classical BPPV, is caused by floating particles within the inner ear. [START_REF] Haynes | Treatment of benign positional vertigo using the semont maneuver: efficacy in patients presenting without nystagmus[END_REF][START_REF] Tirelli | Benign positional vertigo without detectable nystagmus[END_REF][START_REF] Weider | Benign paroxysmal positional vertigo: analysis of 44 cases treated by the canalith repositioning procedure of Epley[END_REF] From the literature, one might have the impression that the so far elusive disorder of sBPPV is a curiosity and affects only few patients with BPPV symptoms.

The first author of this paper (B.B.), who is running a vertigo/dizziness clinic at the General Hospital in Krems, Austria, noted that patients with sBPPV constitute a larger proportion of patients than previously thought. A coauthor of this paper (D.S.), who is running a similar clinic at the University Hospital in Zurich, Switzerland had the same impression. Unlike patients with classical BPPV, patients with sBPPV rarely complain of vertigo in the Dix-Hallpike position, but do experience short vertigo spells during the act of sitting up and for a short time immediately afterwards and sometimes also have abnormal antero-and retropulsion, i.e. oscillation, of the trunk. Moreover, we noted that sitting-up vertigo is typically unilateral, i.e. it occurs after the right or left Dix-Hallpike position, which excludes other mechanisms such as orthostatic dysregulation, changes of intracranial pressure, or increased motion sensitivity.

These observations prompted us to investigate the frequency of sBPPV among patients with dizziness or vertigo and describe the clinical features. In addition, in a representative sample of sBPPV patients, we monitored routine clinical positioning maneuvers by a posturographic table. Specifically, we asked whether patients with sBPPV show increased body sway during sitting up from the Dix-Hallpike position.

METHODS

Frequency of BPPV subtypes

Records of 200 consecutive patients who visited the vertigo/dizziness ambulance at Krems General Hospital, Austria, during the period from January to March 2008 were analyzed. Since most patients had to wait approximately 3-5 months for the appointment, complaints of dizziness / vertigo were generally chronic. The group of patients with benign paroxysmal positioning vertigo (BPPV) was partitioned into three subtypes:

(A) Type 1 BPPV: patients with classical BPPV (B) Type 2 BPPV: patients with subjective BPPV (sBPPV) and short vertigo spells while sitting up (C) sBPPV other than Type 2 BPPV Patients were diagnosed with Type 1 BPPV (= classical BPPV), if the following two criteria were met:

(1) Typical complaints of BPPV (short vertigo when bending forward, lying down, sitting up or turning over in bed) (2) Typical nystagmus provoked by Dix-Hallpike or supine roll maneuvers to either side (as described in Aw et al [START_REF] Aw | Benign positional nystagmus: a study of its three-dimensional spatio-temporal characteristics[END_REF]) Patients were assigned to the Type 2 BPPV group, if the following three criteria were met:

(1) Typical complaints of BPPV (see above) [START_REF] Froehling | Benign positional vertigo: incidence and prognosis in a population-based study in Olmsted County, Minnesota[END_REF] No nystagmus evoked by Dix-Hallpike and supine roll maneuvers (3) Short spell of vertigo during sitting up from either or both Dix-Hallpike positions

The criteria for diagnosing Ménière´s disease included pathologically elevated ratios of summating potential and action potential (SP/AP) measured by non-invasive electrochleography as reported elsewhere (Audiol Neurootol, accepted for publication). The diagnosis of vestibular migraine required normal SP/AP ratio measured by non-invasive electrocochleography and reduction of vertigo episodes under prophylactic treatment (e.g. propranolol, lamotrigine, topiramate).

Posturography

Subjects 20 patients with Type 2 BPPV (11 females; average age: 63 years ± 11 SD) and 9 agematched healthy human subjects were tested after they gave their informed consent. Permission of the local ethical review board was obtained. The patients, apart from signs and symptoms related to Type 2 BPPV, were neuro-otologically normal (no spontaneous nystagmus, no head shaking nystagmus, normal bedside head impulse testing and normal hearing). Cerebral MRI scans of all patients were unremarkable.

Experimental setup

Positioning maneuvers were performed on a table, in which a three-point posturography platform (Posture Evaluating Platform, Med-Eval Ltd, Budapest, Hungary) was incorporated. Signals produced by three mechano-electrical transducers were digitized at 8.33 Hz with 24 bit resolution. Movements of the center of pressure (COP), as recorded by the platform during positioning maneuvers, were analyzed in two dimensions, i.e. the anterior-posterior and lateral directions. The spatial resolution of the calibrated signal was 1 mm.

Paradigms

Subjects were seated on the platform and instructed to avoid unnecessary limb or body movements (with hands, head or body). In sequence, the investigator (B.B.) performed the Dix-Hallpike maneuver on each side and during sitting up the COP movement was recorded. Eye movements were observed by using Frenzel´s glasses and symptoms associated with position changes (vertigo / dizziness, vegetative reactions) were noted. The patients' head was always held by the investigator during positioning, to guide the movement without force. In particular, patients had to sit up without help and special care was taken not to influence the patients' trunk movements during sitting up. In some cases, body oscillations during sitting up were so strong that the investigator had to support the body to prevent falling. In such cases, the trials were excluded from the analysis. In the healthy subjects, Dix-Hallpike maneuvers were done twice to each side and results were pooled to obtain averages. In the patients, however, these maneuvers were carried out four times to both sides in order to assess the effect of repeated positioning. After the first two Dix-Hallpike maneuvers, i.e. one to each side, 90 o supine roll maneuvers were inserted, again one to each side, to assess eye movements and symptoms provoked by stimulation of the lateral semicircular canals.

Data analysis

The COP-curve was windowed. Starting point of the analyzed period was the beginning of the sitting up movement and the analysis ended, when the COP reached the same position as before the Dix-Hallpike maneuver. Hence we usually analyzed a period of around 2-3 seconds during the act of sitting up and immediately afterwards, until the trunk oscillations ended. These were quantified by comparing the windowed original trace to its filtered version obtained by finite impulse response filtering. The filter uses a 2 sec broad moving average filter, which operates by averaging a number of points from the input signal to produce each single point in the output signal. First the average of the first subset of numbers was calculated. The fixed subset was moved forward to the new subset of numbers, and its average as a new value was calculated. The process was repeated over the entire data series. The plot line connecting all the (fixed) averages is the moving average. The traces were windowed and the part containing the movement of sitting up was used. The factor F osc representing trunk oscillations was computed by:

F osc (%) = 100-(L f /L o ) x100
where L o is the length of the original trace and L f the length of the filtered trace measured in mm (see Fig. 2). The original trace, because of its troughs and ridges, is much longer than the filtered trace, which shows an optimal curve that the patients should have produced by sitting up smoothly. The above ratio shows (in percentage) the extent, by which the original trace is longer.

This computation was done for both anterior-posterior and lateral directions of the platform coordinate system. F osc was considered normal within average value ± 2 SD of the healthy control group (between 0 and 14.9 %). For values over the normal limit (pathological high F osc over 14 %) we calculated the asymmetry ratio using a version of the standard Jongkees formula for asymmetry calculations in vestibular testing: [(F osc of side with vertigo -F osc of other side)/( F osc of side with vertigo + F osc of other side)] x 100 We examined 20 patients using posturography. All of them experienced short vertigo spells while sitting up from Dix-Hallpike position, in 7 cases associated with abnormal antero-and retropulsion, i.e. oscillation, of the trunk (Fig. 3). Three of these 7 patients noticed a slight vertigo also in the Dix-Hallpike position on the side of stronger body sway. /* Fig. 3 about here */ In 13 patients with Type 2 BPPV, traces were similar to that of normal individuals (as in Fig. 1A). No transient retropulsion occurred in the anterior-posterior plane and F osc was within the average value ± 2 SD of the healthy control group (under 14.9 %). In this group, 8 patients reported malaise after the forth sitting up maneuver.

RESULTS

Frequency of BPPV subtypes

Posturography

In 7 patients the sitting up movement was interrupted by one or more transient backward movements of the trunk. These retropulsions were the strongest during the initial second of the movement, but lasted as slight, damped oscillations for 1-2 seconds after the patient reached the sitting position. Fig. 4 compares F osc from four consecutive Dix-Hallpike maneuvers to both sides in these 7 subjects. All patients complained of strong nausea and sweating during and after trunk oscillations. There was a clear tendency of decreasing trunk oscillations with each repetition of the Dix-Hallpike maneuver, but the oscillations were always asymmetric, i.e. predominated when sitting up from the right or the left head-hanging position.

/* Fig. 4 about here */ Fig. 5 shows the asymmetry ratio in all seven patients (calculated from the data presented on Table 2. Anterior-posterior and lateral sway during sitting up. Oscillations in the anteriorposterior plane showed reproducible side differences, i.e. were always stronger on the side of subjective vertigo when sitting up. Values for lateral oscillations overlapped between healthy subjects and patients because our analysis proved to be very sensitive concerning lateral body sway and it was difficult even for normal individuals to sit up from the lateral head-hanging position entirely without swinging a little from side to side during the movement. On the other hand, patients did not exhibit extreme body sway in the lateral direction.

Therapy

All 20 patients with Type 2 BPPV were instructed to repeat the Dix-Hallpike maneuver at home, 20 times on both sides (with 4-5 sec. in each head hanging position), once daily, in the morning. We assessed the complaints by a telephone interview after two weeks. By then, none of the patients had complaints. However, some patients described worsening of the dizziness and nausea during the first few days of the exercises. In three patients hospitalization or ambulatory reassessment was necessary, because horizontal canalolithiasis developed. This was treated successfully using the necessary barbecue maneuver. [START_REF] Aw | Benign positional nystagmus: a study of its three-dimensional spatio-temporal characteristics[END_REF] In these three patients, F osc had been normal on both sides before exercises and horizontal canal canalolithiasis occurred on the second day of exercises. Horizontal canalolithiasis developed on the side, on which the patients noticed vertigo when sitting up after Dix-Hallpike maneuver. Patients were instructed to carry on with the training every day and not to quit the exercises. None of the patients have suffered a relapse until the preparation of this manuscript (shortest follow-up 6 months).

DISCUSSION

The main result of our study is that 7 out of 20 patients with subjective BPPV showed increased anterior-posterior body sway, i.e. trunk oscillations. These oscillations could be divided into: 1. short retropulsions during the act of sitting up from the Dix-Hallpike position (patients pushed their body back during the sitting-up movement). 2. damped trunkoscillations for a short time immediately after the patient reached the final sitting position. This is the first time that such retropulsion during sitting-up has been documented in cases of suspected BPPV. Our method was sensitive to the oscillations in the anterior-posterior (diagonal) plane. Lateral trunk sway immediately after the sitting up maneuver was also visible in the patients, but in our limited sample, parameters for lateral sway were not significantly different between patients and healthy subjects. A planned study with a large sample will allow a more detailed comparison of lateral sway between patients and healthy subjects.

Due to the small number of patients, on whom we performed posturographic measurements, detailed statistical significance testing was not possible, but the following trends are noteworthy and should be further studied in larger samples: 1. In 5 of the 7 patients with documented sitting-up trunk oscillations, reproducible differences of trajectories between sitting up from the two sides could be observed. The side of greater retropulsion was also the side on which patients reported more vertigo.

Retropulsion and vertigo decreased with repeated maneuvers

There have been studies to examine body sway using posturography in BPPV cases. However, those studies examined patients with classical BPPV, not subjective BPPV. Still some of the results seem to be relevant for interpreting our results. Stambolieva et al measured the effect of the Epley maneuver in posterior BPPV using classical standing posturography. [START_REF] Stambolieva | Postural stability in patients with different durations of benign paroxysmal positional vertigo[END_REF] Their most important result was, that, even after a successful Epley liberation maneuver, increased body sway remained. Giacomini et al examined twenty patients with canalolithiasis of the posterior semicircular canal and 20 healthy control subjects using static posturography 1 h after diagnosis, 3 days and 12 weeks after the corresponding Epley repositioning maneuver. [START_REF] Giacomini | Long-term postural abnormalities in benign paroxysmal positional vertigo[END_REF] The authors found that patients with BPPV showed, significantly increased body sway both in the lateral and antero-posterior directions compared to healthy subjects. The repositioning maneuver decreased the lateral body sway, while the antero-posterior sway remained, causing the chronic dizziness observed in these patients. In a similar study, Blatt et al found that many patients with BPPV, who had a complete remission of the positional vertigo after treatment, still did not reach normal postural stability; this finding was predominant in elderly patients. [START_REF] Blatt | The effect of the canalith repositioning maneuver on resolving postural instability in patients with benign paroxysmal positional vertigo[END_REF] These studies seem to corroborate our results and to indicate that many patients with classical BPPV continue to suffer from subjective vertigo with postural symptoms as seen in our patients with subjective BPPV. In the study by Giacomini et al this instability could even be assigned to the antero-posterior axis, which may correspond to our finding of sitting-up retropulsion in some of the patients with sBPPV. [START_REF] Giacomini | Long-term postural abnormalities in benign paroxysmal positional vertigo[END_REF] It might be argued that the transient retropulsion observed during sitting up is in fact a sign of cerebellar truncal ataxia. This is unlikely, because the majority of the patients showed a lateralization of the retropulsion (more oscillations when sitting up from one side than from the other side) and sitting-up retropulsion and vertigo swiftly decreased with repetitions of the sitting-up maneuver. The lateralization of sitting-up body oscillation was further quantified by the asymmetry ratio in analogy to Jongkees formula, which is used for calculating vestibular asymmetry after caloric examination. Iwasaki et al already applied such asymmetry calculation for measurements other than caloric reaction. [START_REF] Iwasaki | Ocular vestibular evoked myogenic potentials in response to bone-conducted vibration of the midline forehead at Fz. A new indicator of unilateral otolithic loss[END_REF] Since the patients with subjective BPPV share complaints of classical BPPV, possible etiologies within the labyrinths need to be considered. In his original paper Buckingham has expressed concerns about the fate of repositioned particles. [START_REF] Buckingham | Anatomical and theoretical observations on otolith repositioning for benign paroxysmal positional vertigo[END_REF] He showed on 2 mm thick microsections of human temporal bones that loose otoliths falling from the utricular macula are bound to fall into the short arm of the posterior canal onto the utriculopetal side of the cupula, which opens directly into the inferior portion of the utricle. We hypothesize that these particles may cause chronic Type II BPPV.

Since the vertigo sensation in patients with Type II BPPV can be elicited either in the right anterior-left posterior or in the left anterior-right posterior plane, it is likely that the vertical semicircular canals are involved. More specifically, we suggest chronic short-arm canalolithiasis of the posterior canal on the involved side as a cause of the complaints. In this hypothetical case, in upright posture the posterior cupula is chronically deflected by the weight of the debris that lies on the utricular surface of the cupula. The cupula becomes adapted to the extreme position. Such vestibular adaptation following sustained displacement of the cupula was recently demonstrated by Rabbitt and colleagues (Fig. 6). [START_REF] Rabbitt | Dynamic displacement of normal and detached semicircular canal cupula[END_REF] /* Fig. 6 about here */

The chronic dizziness experienced by Type II BPPV patients may be caused by the constantly changing mechanical load, which the debris exerts on the cupula during everyday activities.

According to the anatomical reconstructions of Buckingham [START_REF] Buckingham | Anatomical and theoretical observations on otolith repositioning for benign paroxysmal positional vertigo[END_REF], when the head is in the hanging position during Dix-Hallpike maneuver, the posterior canal cupula hangs vertically downward, so it will not be deflected further in the Dix-Hallpike position than in upright position, therefore no nystagmus ensues. Moreover, in this head position, the particles fall down from the cupula into the utriculus in the vestibulum. Trunk oscillations during and trunk oscillations shortly after sitting up may be caused by different mechanisms. By losing the mechanical load, and because it had been adapted to an extreme deflected position, the cupula overreacts when, during sitting up, the endolymphatic flow pushes it toward the vestibulum (Fig. 7). Thereby the patients receive the impression that they are falling forward, and transient retropulsion occurs. Trunk oscillation immediately after sitting up may be elicited by particles falling back onto the cupula in the upright position.

/* Fig. 7 about here */ Buckingham may have been right when he suggested that the particles removed from the canals are flushed into the utriculus and then fall on the posterior cupula. Other authors also consider the role of utricular debris. Oas suggested the term "utriculolithiasis" and a new terminology to differentiate "short-arm" from "long-arm" canalolithiasis. [START_REF] Oas | Benign paroxysmal positional vertigo: a clinician's perspective[END_REF] The already cited posturography results of Stambolieva et al and Giacomini et al can also be interpreted in the light of this hypothesis: although positional nystagmus is inhibited by a successful Epley maneuver, the debris floating in the utriculus and settling in the long arm causes increased body sway. [START_REF] Stambolieva | Postural stability in patients with different durations of benign paroxysmal positional vertigo[END_REF][START_REF] Giacomini | Long-term postural abnormalities in benign paroxysmal positional vertigo[END_REF] In our group of 20 patients with Type II BPPV only 7 patients showed transient retropulsions during sitting up. We suggest that in the other 13 cases smaller particles caused less severe symptoms.

BPPV often starts as an acute, severe vertigo spell (with vertigo and vomiting), lasting one or two days, then the symptoms diminish spontaneously over several days, as natural head movements of the patients involuntarily "reposition" particles. The frequency of successful spontaneous reposition depends on which semicircular canals are affected. A spontaneous clearing is easiest in the anterior canal, since the debris may float into the utriculus when the patients assumes a slightly head hanging position and the sits up again. Rolling movements in the bed may cause spontaneous reposition of the horizontal canal. Debris falling into the utriculus may be deposited in the short arm of the posterior canal. Because of its inferiorly positioned and conical form, it funnels remaining, small debris onto the posterior cupula where it remains for a longer time.

Present research on on-site and off-site otolithic growth may answer the question whether otoliths deposited in the short arm of the posterior semicircular canal could persist or even grow there. [START_REF] Lundberg | Assembly of the otoconia complex to the macular sensory epithelium of the vestibule[END_REF] If fine ionic regulations do not function properly, crystal seeds may grow offsite. This may be the case in spite of the fact that under normal conditions Ca 2+ concentration in the endolymph is much below the level required for spontaneous crystallization. [START_REF] Salt | Endolymph calcium increases with time after surgical induction of hydrops in guinea-pigs[END_REF] Ectopic growth is possible because ion-transporting cells are present in the non-sensory parts of the vestibule. [START_REF] Imon | Existence of voltage-dependent Ca2+ channels in vestibular dark cells: cytochemical and whole-cell patch-clamp studies[END_REF] Also, local hypercapnia may increase calcification. Checkley et al, for instance, have shown in fish endolymph that, with constant pH, elevated CO 2 increases CO 3 2concentration and thus accelerates the formation of otolith-aragonite. [START_REF] Checkley | Elevated CO2 enhances otolith growth in young fish[END_REF] In our study, in three patients (out of 20 Type II BPPV patients) a horizontal canal canalolithiasis developed on the second day, after the patients had started everyday training. They developed on the side where the patients noticed vertigo when sitting up after Dix-Hallpike maneuver. In our view this corroborates the hypothesis that the exercises work by dispersing particles that are chronically present in the short arm. During repeated exercises in Dix-Hallpike position they may be repositioned into the utriculus and swim into the horizontal canal.

In conclusion, we suggest that sBPPV is more common than anticipated. Type 2 BPPV may be identical with sBPPV or constitute a major subgroup of it. For a definition of Type 2 BPPV we suggest the following: (1) complaints suggesting BPPV (short episode of vertigo when bending forward, lying down, sitting up or turning over in bed); [START_REF] Froehling | Benign positional vertigo: incidence and prognosis in a population-based study in Olmsted County, Minnesota[END_REF] no nystagmus during either Dix-Hallpike positioning or supine roll to the left and right; (3) short episode of vertigo during and immediately after sitting up from the a Dix-Hallpike position. We did not find patients with sBPPV-complaints who could not be assigned to the group with Type 2 BPPV. Nevertheless, we prefer to leave open the possibility that other mechanisms than chronic short-arm canalolithiasis may cause sBPPV. We suggest that the introduction of Type II BPPV as a valid differential diagnosis of patients with positioning vertigo will lead to better classification and therapy in numerous cases.

FIGURE LEGENDS

Fig. 1. (A, B, C)

A: Movements of the center of gravity in a healthy control subject during the Dix-Hallpike maneuver. The peaks of the trace during movements to and from the head-hanging position are due to the supporting effects of a pillow under the subject's back. The movements after the second peak were analyzed further to assess trunk oscillation elicited by sitting up (see Fig. 2). The arrow shows the time interval of the movement of center of pressure during the act of sitting up from the Dix-Hallpike position. B: Patient 1, Sitting up from the side with vertigo. Posturographic traces of a typical patient with Type II BPPV (Patient 1) during the act of sitting up from the Dix-Hallpike maneuver. When sitting up from the right Dix-Hallpike head-hanging position, the patient's trunk oscillated along the anterior-posterior direction (F osc = 69%), but not when sitting up from the left head-hanging position (F osc =14%). C: Patient 1, Sitting up from the side without vertigo. There is no trunk oscillation. Asymmetry ratio in seven patients (Average + SEM, Patients numbers as in Fig. 4). In case of Patient 5 two points were omitted. These points represented very small F osc values, within the normal range on both sides. Fig. 6 Example of short term adaptation (from Rabbitt et al [START_REF] Rabbitt | Dynamic displacement of normal and detached semicircular canal cupula[END_REF] with permission). The dynamic displacement of the semicircular canal cupula and modulation of afferent nerve discharge were measured simultaneously in response to physiological stimuli in vivo in the oyster toadfish horizontal ampulla. Sustained stimulus caused a decline of the afferent discharge (upper trace) and cupula displacement (middle trace). Elimination of the stimulus (lower trace) therefore resulted in an inhibition of both parameters even below the original baseline (arrows by us). Sustained stimuli sensitize the vestibular organ against stimuli acting in the other direction.

Fig. 1

 1 depicts the movements of the center of gravity in a healthy control subject and in a typical patient (Patient 1) during the Dix-Hallpike maneuver. A typical example of how F osc was determined, is shown in Fig.2. /* Fig.1about here */ /* Fig.2about here */

Fig. 4 .

 4 Fig.5shows the asymmetry ratio in all seven patients (calculated from the data presented on Fig.4.

Fig. 2

 2 Fig. 2Example of the analysis of the posturography curve (Patient 1, same curve as in Fig.1B): the trace has been windowed starting with the deflection marking the beginning of sitting up. Dashed curve: original, continuous line: filtered curve. F osc : 69%. Units: time axis = 1 sec, ordinate = movement of the center of pressure (mm)

Fig. 4 F

 4 Fig.4F osc from four consecutive Dix-Hallpike maneuvers to both sides in seven patients suffering from Type II BPPV with trunkal retropulsion during sitting up from Dix-Hallpike position. Stronger vertigo sensation during sitting up: right side of patients 1,2,4,5,6,7; left side of patient 3. Normal range of F osc : 0-14.9 %; the horizontal line shows the upper limit of the normal range.

Fig. 5

 5 Fig. 5Asymmetry ratio in seven patients (Average + SEM, Patients numbers as in Fig.4). In case of Patient 5 two points were omitted. These points represented very small F osc values, within the normal range on both sides.

Fig. 7

 7 Fig. 7 Hypothetical mechanism of Type II BPPV The right vestibular labyrinth illustrated from medial aspect. Abbreviations: pa = posterior ampulla, psc = posterior (inferior) semicircular canal, hsc = horizontal semicircular canal, asc = anterior (superior) semicircular canal, um = utricular macula. A: The posterior cupula is chronically deflected by the weight of the debris and the cupula becomes adapted to this extreme position B: In the Dix-Hallpike position the particles fall down from the cupula. C: Trunk oscillations during and shortly after sitting up may be caused by different mechanisms. By losing the mechanical load and because of the adaptation the cupula overreacts when, during sitting up, the endolymphatic flow pushes it toward the vestibulum. Thereby the patients receive the impression that they are falling forward, and transient retropulsion occurs. Trunk oscillation immediately after sitting up may be elicited by particles falling back onto the cupula in the upright position.

Table 1 .

 1 Table 1 lists the frequencies of diagnoses of the 200 consecutive patients with vertigo/dizziness seen in the outpatient vertigo center. Type I (classical) BPPV had a frequency of 19.5 %, while Type II BPPV was approximately twice as frequent. All patients with BPPV could be assigned to Type I or Type II BPPV, i.e. there where no patients classified with 'other sBPPV'. Frequency of Type II BPPV among other diagnoses in an unselected group of vertigo/dizziness patients.

	Diagnosis		Frequency (%) Average age (± SD) Males:Females
	BPPV	Type 1 BPPV	19.5	62 ± 14	12:27
		(right: 23, left: 16)
		Type 2 BPPV	43	64 ± 15	36:50
		(right: 49, left: 37)
		other sBPPV	0
	Chronic vestibular insufficiency	5	71 ± 17	4:6
	Vestibular Migraine	8.5	48 ± 14	4:13
	Ménière's disease	15.5	61 ± 12	12:19
	other (phobic vertigo, central	8.5
	neurologic disorders, vestibular
	schwannoma)

Table 2

 2 compares averages (± SD) of F osc between the healthy control group and the group of patients with Type II BPPV.

	F osc ant.-post. (%) F osc lateral (%)

ACKNOWLEDGMENTS

The authors would like to express their thanks to G.M. Halmágyi (Sydney) for his continuing support and useful comments and to two unknown reviewers for their work and suggestions. We thank Dr G. Kronik, Medical Director of Krems General Hospital for his support. Figure 7 was drawn by Zoltán Bodor (www.bodorzoltan.hu) and published with permission of Medicina Kiadó.

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 PMJ and any other BMJPGL products and sub licences such use and exploit all subsidiary rights.

Competing Interest: None declared.


Martina Absinta

MD Maria A Rocca

MD Lucia Moiola

MD Angelo Ghezzi

MD Nicoletta Milani

MD Pierangelo Veggiotti

MD Giancarlo Comi

MD Massimo Filippi
email: massimo.filippi@hsr.it

BRAIN MACRO-AND MICROSCOPIC DAMAGE IN PATIENTS WITH PEDIATRIC MS

Keywords: multiple sclerosis, MRI, pediatric, diffusion tensor, gray matter Brain damage in pediatric MS

Introduction

Although the clinical onset of multiple sclerosis (MS) typically occurs between 20 and 40 years of age, in about 3-12% of cases it can occur before 18 years. [START_REF] Simone | Course and prognosis in early-onset MS: comparison with adult-onset forms[END_REF][START_REF] Banwell | Multiple sclerosis in children: clinical diagnosis, therapeutic strategies, and future directions[END_REF] Following the first clinical episode of central nervous system involvement (CNS), pediatric MS patients typically have a relapsing-remitting (RR) course, with accumulation of fixed disability 15 years or more after the clinical onset. [START_REF] Banwell | Multiple sclerosis in children: clinical diagnosis, therapeutic strategies, and future directions[END_REF] The median time to reach EDSS 3.0 is shorter in patients with adult-onset MS, being 8-12 years. [START_REF] Degenhardt | Clinical prognostic factors in multiple sclerosis: a natural history review[END_REF] The factors related to such a better clinical evolution, at least at short-medium term, of pediatric MS patients in comparison to those with an adult-onset of the disease have not been defined yet.

So far, only a few magnetic resonance imaging (MRI) studies have attempted to define the nature and extent of CNS damage in patients with pediatric MS. Two studies, which recruited exclusively pediatric patients with clinically isolated syndromes (CIS) suggestive of MS, [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF][START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF] found a higher frequency of T2-visible lesions in the brainstem of these patients when compared with adult CIS patients. Magnetization transfer (MT) MRI studies found no abnormalities in the brain normal-appearing white matter (NAWM) and gray matter (GM), as well as in the cervical cord [START_REF] Mezzapesa | A preliminary diffusion tensor and magnetization transfer magnetic resonance imaging study of early-onset multiple sclerosis[END_REF][START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF] of pediatric patients with relapsing remitting (RR) MS in comparison with matched healthy volunteers. Brain DT MRI studies showed that, contrary to what happens in adult-onset MS, [START_REF] Rovaris | Diffusion tensor MR imaging[END_REF] pediatric RRMS patients, compared to matched healthy controls, do not have GM abnormalities and harbor only mild structural abnormalities in the NAWM. [START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF] In order to better characterize the disease-related damage in the brain of pediatric MS patients, we obtained conventional and DT MRI scans from a relatively large population of these patients with either clinically isolated syndromes (CIS) suggestive of MS or RRMS and contrasted their findings with those obtained in a group of age-matched healthy subjects. In addition, to test the hypothesis that CNS involvement in pediatric MS patients is less pronounced than that seen in patients with an adult-onset of the disease, we compared measures obtained in pediatric patients with those of adult patients at similar disease stages.

Patients and methods

a) Patients. We studied consecutively 48 pediatric patients with CIS suggestive of MS (10 subjects) [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] or RRMS (38 subjects) [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF][START_REF] Lublin | Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis[END_REF] and 15 age-matched healthy individuals with no previous history of neurological dysfunction and a normal neurological exam. Patients with acute disseminated encephalomyelitis were excluded according to recently published operational criteria. [START_REF] Krupp | Consensus definitions proposed for pediatric multiple sclerosis and related disorders[END_REF][START_REF] Dale | Pediatric central nervous system inflammatory demyelination: acute disseminated encephalomyelitis, clinically isolated syndromes, neuromyelitis optica, and multiple sclerosis[END_REF] To serve as additional control groups, we also recruited 30 adult CIS patients, [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] and 27 adult RRMS patients. [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF][START_REF] Lublin | Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis[END_REF] Adult patients were selected among the population of MS patients enrolled for research studies during the last two years at our Unit in order to match as close as possible the clinical characteristics of the pediatric MS patients. In addition, to rule out an ageeffect on our results, another group of 18 healthy controls was selected in order to match as close as possible the demographic characteristics of adult MS patients. All CIS patients had paraclinical evidence of disease dissemination in space. [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] Alternative neurological diseases were excluded by appropriate investigations. [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2005 revisions to the "McDonald Criteria[END_REF] The main demographic and clinical characteristics of the six groups of subjects studied are shown in Table 1. Clinical symptoms of CIS were optic neuritis in 2 (20%) pediatric and 8 (27%) adult patients, hemispheric syndrome in 4 (40%) pediatric and 9 (30%) adult patients, brainstem syndrome in 3 (30%) pediatric and 9 (30%) adult patients, and spinal cord syndrome in 1 (10%) pediatric and 4 (10%) adult patients. This brain portion was chosen as these central slices are less affected by the distortions due to B 0 field inhomogeneity, which can affect image co-registration. Additional information about the MR acquisition protocol is given elsewhere. [START_REF] Mezzapesa | A preliminary diffusion tensor and magnetization transfer magnetic resonance imaging study of early-onset multiple sclerosis[END_REF][START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF] c) MRI analysis. T2 lesion volume (LV) of the whole brain as well as of the supra-and infra-tentorial compartments were measured using a local thresholding segmentation technique (Jim 4.0, Xinapse System, Leicester, UK). T2 lesion probability maps (LPMs) were created for each group of individuals. First, a binarized lesion mask from the segmented lesions visible on the T2weighted images was produced. Then, using statistical parametric mapping (SPM2), the non-linear transformation between the T2-weighted images and the Montreal Neurological Institute (MNI)

space was calculated and applied to lesion masks. LPMs were produced by averaging normalized T2 lesion masks (Figure 1). Finally, lesion masks were smoothed with an 8 mm 3 FWHM Gaussian kernel, before their use as input for the statistical analysis.

PGSE images were first corrected for distortion induced by eddy currents using an algorithm which maximizes mutual information between the diffusion un-weighted and weighted images. [START_REF] Studholme | Automated 3-D registration of MR and CT images of the head[END_REF] Then, the DT was calculated, and mean diffusivity (MD) and fractional anisotropy (FA) derived for every pixel, as previously described. [START_REF] Cercignani | Magnetisation transfer ratio and mean diffusivity of normal appearing white and grey matter from patients with multiple sclerosis[END_REF] The diffusion images were interpolated to the same image matrix size as the T2-weighted images, and then the b=0 step of the PGSE scans were co-registered with the T2-weighted images. Using SPM2 and maximum image in-homogeneity correction, [START_REF] Ashburner | Multimodal image coregistration and partitioning--a unified framework[END_REF] brain GM, WM, and cerebrospinal fluid (CSF) were automatically segmented from T2-weighted images. Each pixel was classified as GM, WM or CSF, dependent on which mask had the greatest probability (maximum likelihood) at that location. This generated mutually exclusive masks for each tissue. In each subjects, intracranial volume (ICV) was calculated as the sum of GM+WM+CSF maps. The resulting masks were superimposed onto the MD and FA maps (on which hyperintense lesions were masked out previously), and the corresponding MD and FA histograms of the NAWM and GM were produced. FA histograms were derived only for the NAWM, since no preferential direction of water molecular motion is expected to occur in the GM, due to the absence of a microstructural anisotropic organization of this tissue compartment. For each histogram, the average MD and FA were measured.

d) Statistical analysis. Voxel-wise between-group comparisons of LPMs were performed using SPM2 and an ANCOVA model, including age as a nuisance covariate (p<0.05, family-wise error [FWE] corrected). Using SPSS software, an ANOVA was used to compare structural MRI quantities between: 1) pediatric and adult healthy controls; 2) pediatric healthy controls and pediatric CIS and RRMS patients; and 3) adult healthy controls and adult CIS and RRMS patients.

An analysis of covariance (ANCOVA) adjusted for age and disease duration was used to compare structural MRI quantities between pediatric and adult MS patients. False Discovery Rate (FDR) [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF] was applied to correct for multiple comparisons. The continuous dependent variables with a skewed distribution were log-transformed and tested for normality before ANCOVA was performed.

Normality of the data was confirmed by Shapiro-Wilk test (p>0.05) [START_REF] Shapiro | An analysis of variance test for normality (complete samples)[END_REF]and visual inspection after plotting the histograms. Univariate correlations between clinical findings and structural MR measures were assessed using the Spearman Rank Correlation Coefficient.

Results

All controls had normal brain MRI scans. ICV, whole brain, supratentorial and infratentorial LVs did not differ among patients (Table 2). To take into account head size, an analysis was also run where T2 LV was expressed as a percentage of ICV. Also this analysis did not show betweengroup differences of T2 LV (Table 2). Note: average MD is expressed in units of mm 2 s -1 x10 -3 , FA is a dimensionless index. For statistical analysis: see the text.

Figure 1 shows T2 lesion distribution in the four groups of patients, separately. In all groups, T2-visible lesions were mostly located in the corpus callosum, periventricular, and juxtacortical areas. The between-group comparisons of lesion frequency, showed that in adult RRMS patients lesions were more frequently located in the posterior periventricular WM than in pediatric RRMS patients and in adult CIS patients (p<0.05, FWE corrected) (Figure 2). No difference was found between pediatric CIS patients and the remaining groups. No interaction was found between the factor pediatric/adult and the factor CIS/RRMS.

In Table 2, DT MRI quantities from the six groups of subjects are reported. NAWM and GM DT MRI measures did not differ between pediatric and adult healthy controls (Table 2).

The comparison of DT MRI quantities between pediatric healthy controls and pediatric MS

patients showed a significant between-group difference of NAWM average FA (p=0.004). At posthoc analysis, no difference was detected between pediatric healthy controls and pediatric CIS patients, while pediatric RRMS patients had a significantly reduced NAWM average FA (p=0.003) compared to pediatric healthy controls.

The comparison of DT MRI quantities between adult healthy controls and adult MS patients

showed a significant between-group difference of NAWM average FA (p=0.003), NAWM average MD (p=0.003), and GM average MD (p<0.0001). At post-hoc analysis, no difference was detected between adult healthy controls and adult CIS patients (this difference was significant only when the correction for multiple comparisons was not applied; data not shown). Compared to adult healthy controls, adult RRMS patients had significantly reduced NAWM average FA (p=0.002), and increased NAWM (p=0.01) and GM (p<0.0001) average MD. Compared to adult CIS patients, adult RRMS patients had significantly decreased average lesion FA (p<0.0001), and increased average lesion MD (p=0.006), NAWM average MD (p=0.008), and GM average MD (p<0.0001).

The comparison of DT MRI findings between the four groups of patients, corrected for age and disease duration, showed significant between-group differences of average lesion FA (p=0.001), average lesion MD (p<0.0001), NAWM average MD (0.02) and GM average MD (p<0.0001). At post-hoc analysis, no difference was detected between pediatric and adult CIS patients, whereas adult RRMS patients had significantly higher lesion average MD (p<0.0001) and GM average MD (p=0.009) in comparison with pediatric RRMS ones.

In the whole group of MS patients, significant correlations were found between:

-EDSS vs. age at onset of the disease (r=0. When the analysis of correlations was re-run in the two groups of adult and pediatric patients, separately, all the correlations with T2 LV (including EDSS) remained significant in the adult cohort, while the correlation with GM average MD was not found in the pediatric cohort (data not shown). Similarly, the correlation between average lesion MD and GM average MD remained significant in adult patients only (r=0.40, p=0.002).

Discussion

In this study, we wished to characterize the nature and topographical distribution of T2 lesions, as well as the extent of NAWM and GM damage in patients with pediatric MS in comparison with adult patients at similar disease stages.

Previous studies suggested that the extent and distribution of T2-visible lesions differ between pediatric and adult MS patients. In particular, pediatric CIS patients less frequently than adult CIS patients meet the MRI criteria for disease dissemination in space, [START_REF] Hahn | MRI criteria for multiple sclerosis: Evaluation in a pediatric cohort[END_REF] and show a higher involvement of the posterior fossa, [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF][START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF] especially the pons. [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF] These studies recruited exclusively patients with an initial demyelinating event, within a few months from the onset of the disease, who were studied with conventional MRI only. As a consequence, they were unable to rate the severity of intrinsic lesion damage and to provide a picture of lesion distribution in patients with established MS, likely more representative of the general pediatric MS population.

In our study, the T2 lesion burdens of the whole brain and the supra-and infratentorial compartments, separately, were not significantly different between the two cohorts of pediatric MS patients (CIS and RRMS) as well as between them and the corresponding adult groups. However, the analysis of the regional patterns of lesion frequency revealed a higher occurrence of lesions in the posterior periventricular regions in adult patients with RRMS in comparison with adult CIS and pediatric RRMS patients. Remarkably, we did not find any difference between pediatric patients with CIS and those with RRMS, or between pediatric and adult CIS patients. Several factors might contribute to explain the discrepancy between our findings and those of previous studies. [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF][START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF] First, our pediatric patients were "older" than those of the previous two studies. This might have had an impact on our and previous data since timing of myelin maturation has been considered as a possible explanation of the selective infratentorial involvement in pediatric CIS patients. Second, the time elapsed between the onset of the clinical event in pediatric CIS patients and the acquisition of the MRI scan was more than three months in our study vs. less than 3 months in the study of Waubant et al. [START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF] Since a significant resolution of the initial T2 lesions has been shown to occur in pediatric CIS patients, it is likely that some lesions possibly present at onset were not anymore detectable in our cohort. Finally, all our patients were studied using the same scanner and acquisition protocol, while those of the previous two studies were scanned at different centers and with different sequences. [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF][START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF][START_REF] Filippi | Interscanner variation in brain MRI lesion load measurements in MS: implications for clinical trials[END_REF] Variability of scanners and sequences might have influenced lesion identification and quantification and their distribution on LPMs. [START_REF] Ghassemi | Lesion distribution in children with clinically isolated syndromes[END_REF][START_REF] Waubant | Difference in disease burden and activity in pediatric patients on brain magnetic resonance imaging at time of multiple sclerosis onset vs adults[END_REF][START_REF] Filippi | Interscanner variation in brain MRI lesion load measurements in MS: implications for clinical trials[END_REF] Clearly, considering the relatively small number of pediatric CIS patients we recruited, we can not completely rule out that our study was underpowered for detecting any significant difference between pediatric CIS patients and the remaining groups. Nevertheless, the demonstration that adult RRMS patients have a higher occurrence of lesions in the posterior periventricular WM, where several "clinically eloquent" pathways run, and the demonstration that infratentorial structures are similarly involved in all patients (Figure 1) fit with the notion of a favourable disease outcome in this latter group.

We also quantified the extent of microscopic damage in lesions, NAWM and GM by means of DT MRI, which has been shown to be sensitive to disease-related changes both in adult and pediatric MS patients. [START_REF] Mezzapesa | A preliminary diffusion tensor and magnetization transfer magnetic resonance imaging study of early-onset multiple sclerosis[END_REF][START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF][START_REF] Rovaris | Diffusion tensor MR imaging[END_REF] Noteworthy, in a preliminary study, [START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF] which included part of the pediatric patients of the present analysis, we did not find DT MRI changes in the brain GM of pediatric RRMS patients when contrasted to matched healthy controls. The previous study was limited by the small sample of pediatric patients enrolled [START_REF] Tortorella | MRI quantification of gray and white matter damage in patients with early-onset multiple sclerosis[END_REF] and by the fact that pediatric MS findings were not compared with those from adult patients. The present result are, therefore, important, since they provide a compelling evidence for a sparing of brain GM in pediatric-onset MS. Indeed, we found no GM abnormalities in both CIS and RRMS patients in comparison with healthy controls. In addition, GM average MD was significantly higher in adult RRMS patients in comparison with the corresponding pediatric group. Diffuse damage to the brain GM has been

shown virtually in all adult MS patients with different disease courses. [START_REF] Cercignani | Magnetisation transfer ratio and mean diffusivity of normal appearing white and grey matter from patients with multiple sclerosis[END_REF][START_REF] Bozzali | Quantification of brain gray matter damage in different MS phenotypes by use of diffusion tensor MR imaging[END_REF][START_REF] Ceccarelli | A voxel-based morphometry study of grey matter loss in MS patients with different clinical phenotypes[END_REF][START_REF] Chard | Brain metabolite changes in cortical grey and normal-appearing white matter in clinically early relapsing-remitting multiple sclerosis[END_REF][START_REF] Ge | Magnetization transfer ratio histogram analysis of gray matter in relapsing-remitting multiple sclerosis[END_REF] The relative preservation of GM integrity in pediatric MS is in line with the recent demonstration, using voxelbased morphometry, of a selective atrophy of the thalamus in these patients, with sparing of the cortical GM and of the remaining deep GM nuclei. [START_REF] Mesaros | Evidence of thalamic gray matter loss in pediatric multiple sclerosis[END_REF] It also fits with the results of a functional MRI study of the motor system in pediatric MS, which showed a maintenance of a selective and strictly lateralized pattern of brain activations, thus supporting the notion of a preserved functional reserve in pediatric MS that might be the result of a structural GM integrity. [START_REF] Rocca | Is a preserved functional reserve a mechanism limiting clinical impairment in pediatric MS patients?[END_REF] Recently, the introduction of double inversion recovery (DIR) sequences has allowed to demonstrate macrocopic GM lesion in the majority of the adult MS phenotypes. [START_REF] Geurts | Intracortical lesions in multiple sclerosis: improved detection with 3D double inversion-recovery MR imaging[END_REF][START_REF] Calabrese | Detection of cortical inflammatory lesions by double inversion recovery magnetic resonance imaging in patients with multiple sclerosis[END_REF][START_REF] Calabrese | A three-year MRI study of cortical lesions in relapse-onset multiple sclerosis[END_REF] These GM lesions have been related

to the accumulation of widespread GM damage, in terms of atrophy, and to the development of irreversible disability. [START_REF] Calabrese | A three-year MRI study of cortical lesions in relapse-onset multiple sclerosis[END_REF][START_REF] Calabrese | Cortical lesions in primary progressive multiple sclerosis: a 2-year longitudinal MR study[END_REF] Unfortunately, we did not acquire DIR sequences in our patients, as a consequence we can not exclude that a reduced amount of GM macroscopic lesions might be among the factors responsible for the different GM DT MRI findings between our pediatric and adult cohorts.

Another intriguing finding of this study derives from the analysis of NAWM involvement, which showed no DT MRI changes in the CIS cohorts (independently of the age at onset), and a similar extent of DT MRI changes (reduction of FA and increase of MD) in the RRMS forms of the disease. The finding of a relatively mild NAWM involvement in CIS patients agrees with the results of previous studies in adult patients, which demonstrated absent or only subtle NAWM abnormalities. [START_REF] Pulizzi | Determinants of disability in multiple sclerosis at various disease stages: a multiparametric magnetic resonance study[END_REF][START_REF] Gallo | Diffusion-tensor magnetic resonance imaging detects normal-appearing white matter damage unrelated to short-term disease activity in patients at the earliest clinical stage of multiple sclerosis[END_REF] The correlation found between NAWM DT MRI abnormalities and the extent of T2-visible lesions suggests that Wallerian degeneration of axons passing through focal, macroscopic lesions is likely to be one of the factors contributing to NAWM damage in this disease.

Finally, we also quantified the extent of intrinsic damage to T2-visible lesions using DT MRI. Such an analysis showed a higher lesion average MD in adult than in pediatric RRMS patients, which might be a reflection of a better capability of the "young" brain to react to the inflammatory process through a more efficient tissue repair. Remarkably, in the whole cohort of patients and in the adult cohort separately, average lesion MD was correlated with MD changes in the GM, while such a correlation was not found in pediatric patients. These results suggest that the severity of damage of macroscopic T2-visible lesions might be an additional factor contributing to the observed GM changes in adult MS patients in comparison with the pediatric group.

Figure 1 .

 1 Figure 1. Representative slices showing lesion probability maps, superimposed on a T1-weighted

Figure 2 .

 2 Figure 2. Areas of significant increased lesion occurrence in adult patients with relapsing remitting

Table 1 .

 1 Main demographic and clinical characteristics of the six groups of subjects studied.

		Pediatric	Adult	Pediatric	Pediatric		Adult
						Adult CIS
		healthy	healthy	CIS	RRMS		RRMS
						patients
		volunteers	volunteers	patients	patients		patients
	Number of subjects	15	18	10	38	30	27
	Women/Men	10/5	11/7	6/4	24/14	21/9	18/9

the second-last caudal slice positioned to match exactly the central slices of the other image sets.

Table 2 .

 2 Conventional and diffusion tensor MRI findings in the six groups of subjects studied.

	Pediatric	Pediatric	Pediatric	Adult
	Adult healthy		Adult CIS
	healthy	CIS	RRMS	RRMS
	volunteers		patients
	volunteers	patients	patients	patients

Acknowledgement/Funding. This study was partially supported by a grant from Fondazione Mariani (R07-62).

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Competing Interest: None declared.