# Transbulbar B-Mode Sonography for Clinical Phenotyping Multiple Sclerosis
## 2 Materials and Methods  
In an unblinded, comparative, cross-sectional study, we investigated 41 (20 males, 21 females) relapse-free RRMS patients and 19 (8 males, 11 females) sex-matched SPMS patients and 35 (17 males, 18 females) healthy controls subjects, making a total of 95 eyes. We defined as \"relapse-free\" patients which are free from clinical (neurological worsening) or radiological (increasing MRI-detected lesion load) relapse by at least three months. Given the negligible inter-side difference [8], the right eye of each subject was investigated by TBS as part of their clinical examination. Within 24 h, brain MRI was conducted and visual evoked potentials were measured among the MS group. Transbulbar B-mode sonography was carried out personally by the primary author of this work, a neurophysiologist certified by the Italian Neurosomology Society (Italian Society of Neurosonology and Brain Haemodynamics (SINSEC)). A GE Vivid 7 ultrasound system (GE Vingmed Ultrasound AS, Horten, Norway) was used in accordance with [PERSON] et al. [1; 2]. Briefly, with the 4-11 MHz linear probe placed on the temporal part of the closed upper eyelid, the optic nerve was depicted in a transverse plane, revealing the papilla and the optic nerve in its longitudinal course at 3 and 5 mm behind the eyeball bilaterally. The distance between the external borders of the hyperechoic area surrounding the ON was quantified as the ONSD. P100 latency (P100L) and amplitude (P100A) were calculated in the right eye at a distance of 1 m from the checkboard, in accordance with [PERSON]'s pattern-reversal protocol. Briefly, full-field visual evoked potentials (VEPs) were extracted during 1 Hz alternating standard pattern stimulus on a high-contrast black and white chequerboard with a square element size of 1 \(\pm\) 20\({}^{\circ}\) per side, after one hundred stimuli. Given that mean of P100L and P100A both exceeding significantly their normative data, no healthy control group was planned. Specifically, values of 98.19 \(\pm\) 0.75 ms and 7.23 \(\pm\) 2.16 \(\upmu\)v were used as normal cut-off for P100L and P100A, respectively [14]. All patients were positioned and imaged with a 1.5 T Philips MR apparatus (180 mT/m) (Achieva, Philips Medical Systems, Best, The Netherlands), in accordance with international guidelines [15]. SENSE (Sensitivity Encoding) parallel imaging method with contrast enhancement (Gadovist single dose, 10 min post-administration) was used. Modalities and acquisition sequence types are summarized in Table 1.  
The MRI post-analysis was conducted using \"Sienax\" software (Analysis Group, FMRIB, Oxford, UK) to determine brain parenchymal fraction (BPF), \"MIPAV\" (National Institutes of Health Center for Information Technology Rockville, MD, USA) to calculate the T1 and T2 lesion loads (T1 LL, T2 LL) and \"SPM12\" (Functional Imaging Laboratory, Wellcome Trust Centre for Neuroimaging, Institute of Neurology, London, UK) to calculate total brain volume (TBv), grey/white matter volume (GMv, WMv), CSF ventricular volume (CSFv), and peripheral grey matter volume (PGMv). The clinical parameters considered were EDSS score and sub-scores and disease duration (DD). Developed by [PERSON] in 1983, the EDSS is a universal scale for quantifying neurological impairment in MS patients [16]. This method numerically quantifies overall disability with reference to the eight functional systems (pyramidal, sensitive, cerebellar, brain-stem, etc.) and allows neurologists to assign a functional system score (the so-called functional sub-score) to each of them. Co-morbidity, including ophthalmic and toxic-deficiency diseases, entailed exclusion from this study. Patients that relapsed three months before the study and those affected by retrobulbar optic neuritis were also excluded. We also considered the ratio of ONSD at 3 mm from the eyeball (ONSD3) to ONSD at 5 mm from the eyeball (ONSD5), also called the myelination index (MI) [8]. We used the MI to associate knowledge of ONSD with the thickness of the myelin coating of the ON.