Evaluation of Axonal Optic Nerve Damage Using Visual Evoked Potentials and Optical Coherence Tomography in Patients With Multiple Sclerosis

in MS is not mostly related to demyelination, but to an axonal and neuronal loss (1–4). The necessity for the closer analysis of axonal tissue arises from the fact that there is a growing need to develop biomarkers for monitoring the disease progress and modeling the prognosis as well as the evaluation of treatment effectiveness (5). An acute episode of optic neuritis (ON) is observed in 30%–70% of MS patients during the course of the disease (6, 7). The damage of vision pathways is an ideal model for neurodegenerative processes in MS (8), which theoretically might


Introduction
disease of the central nervous system that often affects young patients and leads to a progressive neurological defect.Relatively recent studies have conin MS is not mostly related to demyelination, but to an axonal and neuronal loss (1)(2)(3)(4).The necessity for the closer analysis of axonal tissue arises from the fact that there is a growing need to develop biomarkers for monitoring the disease progress and modeling the prognosis as well as the evaluation of treatment effectiveness (5).An acute episode of optic neuritis (ON) is observed in 30%-70% of MS patients during the course of the disease (6,7).The damage of vision pathways is an ideal model for neurodegenerative processes in MS (8), which theoretically might coherence tomography (OCT) captures images from the retina (9), the only place in a human body where the investigation of the axonal tissue layer is possible in vivo regardless of the myelin sheath condition.It has repeatedly been shown that damage to the retinal can be detected even in patients without a history of an acute episode of ON, suggesting of subclinical and progressive damage of the afferent visual system (10)(11)(12)(13).A neurophysiological method -visual evoked potentials (VEPs) -has already been used for the evaluation of axonal optic nerve damage for many years.This method was also mentioned in the McDonald's criteria used for the diagnostics of MS in 2001 and 2005 (14,15).The loss of axonal tissue is characterized by changes in the VEP amplitude, but the prolongation of latency refers to optic nerve demyelination (16).The aim of our study was to analyze the clinical utility of VEP and OCT in the diagnosis of optic nerve axonal damage and to determine the correlation between the RNFLT thickness, which is considered a structural parameter of the retina, and the VEP amplitude, a functional visual parameter, in MS patients with and without a history of acute ON.

Material and Methods
The cross-sectional study included 69 relapsingremitting MS patients with and without a history of ON, recruited from the Multiple Sclerosis Center at Pauls Stradins Clinical University Hospital.A history of ON was documented based on symptoms and clinical signs (17).However, not all patients had a documented episode of ON.The study involved months before examinations.The mean time after an episode of ON was 19.2 months.Patients were not given corticosteroid therapy for at least 30 days before the study, while some patients continued immunomodulatory treatment.Patients suffering from neurological diseases other than MS and ophthalmological or systemic ones that could affect the afferent visual system were not included into the study.MS diagnosis was established by a neurologist according to the McDonald's criteria.A neurological examination with impairment assessed using the Kurtzke Expanded Disability Status Scale (EDSS) was performed.The EDSS is based on data from the neurological examination and patient's ability to walk.Scores range from 0 (indicating no neurologic impairment) to 10 (death due to MS).An experienced ophthalmologist performed a complete ophthalmologic examination: determination of visual acuity, measurement of ocular tension, biomicros-All the patients underwent the pattern-reversal VEP stimulation in a dark and quiet room for both eyes by using the RETI-port 21 (Roland Consult, Germany).Subjects were seated at a distance the gaze at the red dot in the center of the screen.Full correction of refraction was performed if necessary.Active and reference gold disc electrodes were placed at Oz and Fz, respectively, according to the international 10-20 system.The monocular stimulation of vision with the black-white video monitor at a frequency of 1.6 Hz was performed, and stimulus patterns were presented on a video screen subtending 12×15 degrees of visual angle with a pattern and analyzed by means of repeated (100) stimulations 2 times for each eye.N75/P100 amplitudes were measured.The VEP amplitude was evaluated based on the laboratory-designed reference value, to assume amplitude under 8.2 μV as an abnormal one.
The RNFLT thickness in 6 standard sectors (temporal, temporal upper, temporal lower, nasal, nasal upper, and nasal lower) was measured for all subjects on OCT (Heidelberg Engineering SPEC-TRALIS) using the TruTrack TM active eye-tracking system.All the scans were performed by the same experienced ophthalmologist.Poor-quality scans were excluded.The RNFLT thickness in the reference range was represented by the green background.The presence of the defect was determined based on the machine normative database; a red col-The patients with MS were divided into 2 groups.history of unilateral ON.The second group comprised 44 patients without a history of ON.The control group consisted of 14 age-matched individuals.
Descriptive statistics were used for data presentation and analysis.The chi-square test or the Fisher exact test was used to evaluate qualitative variables.Quantitative variables for both the groups were analyzed using the Student t test.Correlation was assessed by the Spearmen correlation.The receiver operating characteristic (ROC) curve and the area under the curve were analyzed.The MedCalc 12.0 differences between the areas under ROC curves. P<0.05.

Results
The study included 25 eyes affected by ON, 25 contralateral, unaffected eyes of the same group, and 88 eyes of MS patients without a history of ON.The age of the patients ranged from 17 to 65 years.
ing the study groups (t=1.90,P=0.06).The mean EDSS score of MS patients was 1.94.The disease duration ranged from 0 to 400 months.The clinical and demographic characteristics of the patients included in the study are summarized in Table 1.
Table 2 shows the comparison of the mean VEP amplitude between MS patients with and without a history of ON. was found between the RNFLT thickness and the mean VEP amplitude in the MS patients with a history of ON (r s =0.42, P=0.01) as well as in the MS patients without a history of ON (r s =0.44, P<0.001).
The abovementioned relationships are depicted in served in the control group (P=0.47).
A separate analysis of the MS patients' population with a history of ON revealed an abnormally reduced RNFLT thickness in 16 ON-affected eyes (64%), while a decrease in the mean VEP amplitude was observed in 18 cases (72%).
Similar results were obtained studying the other ON-unaffected eye of the same patients' population, to assess a subclinical optic nerve involvement.The reduced RNFLT thickness was found in 6 eyes (24%) and the decreased mean VEP amplitude in 11 cases (44%).
By using the combination of both methods in the ON-affected eyes, abnormalities were detected in 13 cases (52%).However, regarding the ON-unaffected eyes, optic nerve damage was documented in 4 eyes (16%).
While seeking for an answer to the question how high the possibility is to make mistakes by using only one method, it turned out that in the ONaffected eyes with a normal mean VEP amplitude, (12%), while in case of the normal RNFLT thickness, the average VEP amplitude was reduced in 5 cases (20%).Similarly analyzing the other ON-unaffected eyes, the average VEP amplitude was normal with an abnormal RNFLT thickness in 2 cases (8%), while 7 cases (28%) had a reduced average VEP amplitude with the normal RNFL thickness.
It was observed that between ON history and presence of a reduced VEP amplitude there exists P=0.04), and such relevance was approved also between the history of ON and the presence of the decreased RN-FLT thickness (P=0.03).
Referring to the ROC curve analysis and setting the clinical ON signs as the gold standard, the sencase were 64% and 90%, respectively, with the diagnostic accuracy being 74%.Similarly, measuring the RNFLT thickness and setting the ON clinical signs as the gold standard, the corresponding values of OCT were 50%, 100%, and 73%, respectively.The positive predictive value of the RNFLT thickness measured by OCT was greater than that of the VEP amplitude (Table 3).

Discussion
Similar to previous studies (11-13, 18, 19), our damage in ON-unaffected eyes can be diagnosed by using the measurements of both the VEP amplitude and the RNFLT thickness.These results support the need to apply these tests to MS patients even without a history of ON in order to get the picture of axonal optic nerve lesions.In addition, both of these methods are useful in equivocal clinical situations, when the symptoms of ON are weakly expressed or the patient is not able to assess them adequately due to cognitive impairment.
Relying on the data obtained, our study showed an equally strong correlation between the mean VEP amplitude and the RNFLT thickness both in the eyes with or without a history of ON.In addition, our results indicated a correlation between the reduced VEP amplitude and decreased RNFLT thickness in the patients with a history of ON.This concurs with the data in literature and supports the use of these 2 methods to detect ON.Both the measurements of the RNFLT thickness by OCT and the VEP amplitude in the AUC analysis are equally qualitative parameters for the diagnostics of optic nerve damage due to ON.However, using the OCT method, optic nerve damage was diagnosed in fewer cases as compared with the VEP method both in the eyes affected and unaffected by ON. sensitivity by 14% lower as compared with VEP, and it was lower than that reported in the literature (18).Nevertheless, OCT had a higher positive predictive value, which can be explained by well-designed cutoff values in the device normative database.One of the explanations for the low sensitivity of OCT could be the fact that only the anterior visual pathway can be assessed by this test, meanwhile the VEP can provide an insight into the integrity of both the anterior and posterior visual pathways.Moreover, it should be taken into account that after an ON episode, axonal damage could be secondary to demyelination, but the reduction in the RNFLT thickness proceeds retrograde and is therefore not always complete.To improve the sensitivity of OCT, some authors (20)(21)(22) have recently offered the measurements of the neuronal ganglion cell layer and the inner plexiform layer, which should indicate axonal damage more accurately than the measurements of the RNFLT, as these structures contain no additional glial tissues and blood vessels (23).Mistakenly thicker RNFLT could also be caused by possible glial tissue replacement for axonal tissue loss, which is included in the total RNFLT thickness.However, it should be noted that the measurements of the ganglion cell neuronal layer and the inner plexiform layer are possible only through manual segmentation, which is a relatively time-consuming process and therefore may be hardly ever applied in clinical practice.

Conclusions
The obtained results suggest that despite a sigand the VEP amplitude, the diagnosis of both clinical and subclinical optic nerve damages is made more convincingly by using VEP.From this conclusion it follows that if circumstances require using a single method for the approval of an ON history, VEP is more preferred.However, the combination of the VEP and OCT methods would facilitate the

Statement of Conflict of Interest
r s =0.42, P=0.01 O r s =0.44, P<0.001 the temporal quadrant (RNFLT) thickness and average visual evoked potential (VEP) amplitude in ON diagnostics using ROC curves