From the point of view of pathogenesis, multiple scle-rosis (MS) has long been regarded as an inflammatorydemyelinating disease, the primary target of the destructiveprocess being the myelin sheaths of neurons and oligoden-drocytes (oligodendropathy). Basic studies in recentdecades have shown that MS also involves damage to theaxons and bodies of neurons, the severity of this damagevarying in intensity at different lesions both within a givenpatient and in different MS patients [6, 20]. The mechanismof nerve cell damage in MS remains incompletely under-

stood. The literature lacks a consistent view of the develop-ment of the neurodegenerative process in this disease [9].

Difficulties in assessing neurodegeneration are largelyexplained by the lack of standardized methods for evaluat-ing this process in MS and clear concepts of the timeframes, distribution, and mechanisms of neurodegenerationin this disease. Thus, the question of the appropriateness ofthe methods used for assessment of the activity of the neu-rodegenerative process in MS is currently relevant [2].

Many investigators now recognize that the leading rolein increases in neurological and cognitive deficits in MS isplayed by diffuse degenerative processes in the “apparentlynormal white matter” (ANWM) and “apparently normalgray matter” (ANGM) in both the brain and spinal cord. Theintensity of global axon damage in the ANWM does not cor-relate with the quantity, size, or location of focal lesions inthe brain [19] and spinal cord [10]. Pathomorphological data

Neuroscience and Behavioral Physiology, Vol. 43, No. 8, October, 2013

The Neurodegenerative Process in Multiple Sclerosis and the Possible Neuroprotective Effect of Treatment with b-Interferon 1a (Avonex)

M. V. Davydovskaya,1 A. N. Boiko,1 A. E. Podoprigora,2

I. N. Pronin,2 V. N. Kornienko,2 and E. I. Gusev1

0097-0549/13/4308-0907 ©2013 Springer Science+Business Media New York

907

Translated from Zhurnal Nevrologii i Psikhiatrii imeni S. S. Korsakova, Vol. 112, No. 7, pp. 36–41, July, 2012.

The dynamics of changes in the N-acetylaspartate/creatine ratio (NAA/Cr) in brain tissue in patients withmultiple sclerosis (MS) were studied by proton multivoxel magnetic resonance spectroscopy (PMRS) toidentify relationships between these parameters and the clinical status of patients to assess the potential forusing this method in monitoring disease severity and the extent of the neurodegenerative process. The poten-tial neuroprotective effect of treatment with an interferon β-1a formulation for intramuscular administration(IFNβ-1a, Avonex) in patients with remitting MS (rMS) was also studied. A total of 26 patients with rMStook part in the study. Investigations included history-taking, neurological examination with assessment onthe EDSS, neuropsychological testing, and dynamic MRI studies using PMRS. A decrease in the NAA/Crratio was seen in patients with rMS as compared with healthy subjects. Analysis of the NAA/Cr ratio at oneyear demonstrated a significant reduction. A negative relationship was found between the NAA/Cr ratio inbrain tissue and the levels of neurological and cognitive deficit. Analysis of intramuscular IFNβ-1a treatmentof MS demonstrated a stabilizing neuroprotective effect through the one-year treatment period, which maybe associated with the effects of this agent on the neurodegenerative process in MS.

Keywords: multiple sclerosis, neurodegenerative process, proton magnetic resonance spectroscopy, interferon β-1a forintramuscular use.

1 N. I. Pirogov Russian National Medical Research University.2 Department of Radiological and Surgical Methods for

Diagnosis and Treatment, Academician N. N. Burdenko Research Institute of Neurosurgery, Russian Academy ofMedical Sciences, Moscow; e-mail: das456@yandex.ru.

are consistent with data from MRI spectroscopy studies ofmetabolites in the CNS [11]. This leads to the conclusionthat diffuse axon damage is only partly determined by pro-cesses occurring in the corresponding foci.

Proton magnetic resonance spectroscopy (PMRS) is anoninvasive method for vital assessment of the level of neu-ron damage [5, 12], which provides assessment of qualitativeand quantitative biochemical changes occurring in brain tis-sue [5]. Metabolic changes on MR spectrograms are apparentas peaks reflecting changes in several metabolites, of whichthe best studied are N-acetylaspartate (NAA), creatine (Cr),choline, myoinositol, and the lactate-lipid complex.

NAA is an aminoacyl derivative synthesized in neu-rons and transported along axons [27]. It is located in neu-rons and axons in the brain in adult humans [7, 29].Antibodies to NAA stain only neurons, leaving glial cellsunstained [23]. Complete degeneration of the optic nerve(ON) in rats after nerve transection is accompanied by com-plete loss of NAA with persistence of oligodendroglial cells[31]. Comparison of PMRS data with histological studies ofbiopsy specimens has also provided evidence that thismetabolite characterizes nerve cell status [8]. Changes inNAA levels are regarded as a marker of the state of CNSnerve cells; in particular, decreases in NAA reflect theseverity of axonal dysfunction or loss [27]. NAA levels areevaluated either as absolute values or, better, in relation tocreatine, as the NAA/Cr ratio [24]. A number of studieshave now been undertaken indicating that multivoxelPMRS is an informative method for detecting focal and dif-fuse changes in brain tissue in MS [16, 24, 26, 28].

Various data show that axon and neuron damage in MScan be detected from disease onset, or as a clinically isolatedsyndrome (CIS) [13], with subsequent continuous progres-sion; some investigators [9, 30] believe that this has primaryresponsibility for the accumulation of irreversible neurolog-ical deficit. This makes the important role of neuroprotectivetherapy in the treatment of MS understandable.

The dominant position among methods for the patho-genetic treatment of MS is currently occupied by the use ofinterferon-β (IFNβ) formulations and glatiramer acetate(GA), i.e., MS course-modifying agents (MSCMA), whichare used as first-line agents. These therapeutic agents havebeen found to be able to decrease the frequency and severi-ty of exacerbations and to slow the rate of accumulation of

neurological deficit, which is supported by improvementsseen in brain and spinal cord MRI scans [14, 15]. However,it should be emphasized that MSCMA allow the activity ofthe pathological process to be controlled, especially at theearly stages of disease, though progression cannot bestopped [1]. No treatment method completely able to termi-nate progression of MS has yet been developed. The maintarget for the actions of contemporary MSCMA is autoim-mune inflammation. The mechanisms of the possible directand indirect neuroprotective effect of this treatment arebeing studied [3, 4].

The aims of the present work were to study the devel-opment of the neurodegenerative process in remitting MS(rMS) using PMRS and to assess the neuroprotective poten-tial of the treatment of MS with IFNβ formulations.

Materials and MethodsPatients with MS under review at the Moscow City

Multiple Sclerosis Center (MSMSC) from 2008–2011 werestudied.

During the period of one year, 26 patients with rMSdiagnosed in accordance with the MacDonald criteria (2005)were observed; patients were 18–52 (mean 32.31 ± 9.3) yearsold. Clinical characteristics are presented in Table 1.

Patients’histories were analyzed retrospectively. Clinicalinvestigations included assessment of status on the disabilityscale (EDSS) and the functional systems scale (FS), whichis based on the Kurtzke scale [18]. All patients with MS alsounderwent testing to assess the presence of cognitive dys-function (MMSE) and quantitative evaluation of the abilityto switch from one type of activity to another, i.e., the Stroopverbal color test and the Paced Auditory Serial Addition Test(PASAT); the Beck depression scale and chronic fatigue scale(MFIS) were also used.

The control group consisted of 37 patients (16 men,21 women) reviewed at the MCMSC with suspected onsetof MS but found not to have this disease. The mean age ofpatients in the control group was 34.9 ± 11.3 years (range22–56 years). Most rMS and control patients had highereducation.

Prior to the study, patients had not received immunomo-dulatory treatment with MSCMA (first presentations to theMSMSC, termination of treatment due to inefficacy, sideeffects, or unwillingness to take immunomodulatory therapy).

Davydovskaya, Boiko, Podoprigora, et al.908

TABLE 1. Clinical Characteristics of MS Patients and the Control Group

In an open, observational study, 10 patients with rMSreceived, in accordance with indications, immunomodulatorytreatment in the form of interferon β-1a (IFNβ-1a) at a stan-dard dose of 30 μg i.m. once weekly. Patients were includedin the treatment group in compliance with international andRussian guidelines for the use of MSCMA. By decision ofthe Special Committee of the Moscow Health Department,the agent is approved for MS patients aged 18 years or olderwithout any other neurological disease to cause their symp-toms and without any contraindications to use of the agent.

The patients treated with IFNβ included six womenand four men aged 22–43 (mean 32.1 ± 7.4) years. Onset ofMS was at age 20–37 (mean 27.1 ± 3.5) years. At themoment of recruitment into the study, disease duration was3–11 (mean 5.4 ± 2.95) years. During the year before treat-ment, the mean frequency of exacerbations was 1.6 ± 0.46per year. At the beginning of the observations, before treat-ment, EDSS was 1.0–3.0 (mean 1.75 ± 0.59) points.

All patients underwent MRI scans and PMRS twice dur-ing the observation period (at the beginning of the study andat least one year later). Combined MRI/PMRS studies of thebrain were performed in a single session using a SIGNAEXCITE (GE Healthcare) magnetic tomograph with a mag-netic field strength of 1.5 T. The study program consisted ofobtaining axial T2-weighted images, T2-WI (TR/TE == 6800/90) and PD-WI (TR/TE = 6800/10), with slice thick-ness = 3 mm, T1-WI (3D-SPGR, TR/TE = 20/4), with slicethickness = 1.5 mm before and after intravenous administra-tion of a standard dose of contrast medium (0.2 ml/kg bodyweight). Pathological foci (hyperintense on T2-WI, hypo- orisointense on T1-WI, with or without accumulation of con-trast medium by foci on post-contrast T1-WI) were assessedby an independent expert who had no information on clini-cal pattern of disease. Foci were assessed on routine MRIscan tomograms for all slices, not only in the “zone of inter-est” (ZI), and these were used for further PMRS analysis.

In the T2-WI series, slices including the maximum volumeof corpus callosum and white matter and generally contain-ing one or several pathological foci were selected. Studieswith proton multivoxel MR spectroscopy were performedat the selected level (PMRS, 2D CSI): TR/TE = 1000/44,16 phase coding steps, voxel thickness = 10 mm, CSI voxelvolume = 2.25 cm3, ZI area was the maximum possible forcapturing the greater volume of brain matter on the slice.PMRS data were processed using the integrated Functoolprogram for quantitative assessment with determination ofthe ratios of several basic metabolites (mI, Cho, Cr, NAA,and LL) in each voxel, including in lesion foci and in visu-ally undamaged matter. Relative concentrations of metabo-lites in individual voxels were determined relative to the Crreference peak and mean values for the whole ZI were thencalculated to determine mean ratios of metabolites for thewhole study (Fig. 1). The size and location of ZI remainedconstant for each patient on dynamic observations. Thus,this method provided dynamic observation of metabolitelevels in the large central ZI in the brain at the level of thecorpus callosum, including both foci of demyelination andvisually undamaged white matter (ANWM).

MRI/PMRS studies were performed outside exacerba-tions of MS; when exacerbations occurred by the time spec-ified by the protocol, MRI/PMRS was deferred to six weeksafter clinical improvement. In the control group, PMRS wasperformed with five patients (three women and two men)without neurological disease and with ages and genderscomparable to those of the study group. Mean age was30.33 ± 6.8 years.

Results were analyzed statistically in IBM SPSS Statis-tics 19.0. Differences were regarded as significant at p < 0.05.

Results and DiscussionResults of psychological and psychiatric testing of MS

patients. Before observations, none of the patients had clearsigns of depression (0–19 points, mean 7.0 ± 5.9 on theBeck depression questionnaire); scores on the chronicfatigue scale were less than 36 points. Patients’ scores onthe MMSE were 26 or more points. There were no changesin test performance on repeat testing at one year. Thus, therewere no patients with dementia in the study group.

At the beginning of the study, the mean number of cor-rect responses in the PASAT test was 48.8 ± 8.0 (29–58 cor-rect responses, p = 0.003 compared with the control group).On repeat testing at one year, there was a significantdecrease in the number of correct responses – to a mean of46.5 ± 9.0 (26–58 correct responses, p = 0.01, Wilcoxontest). The results from this test showed a decrease in infor-mation processing speed in MS, with impairment to short-term memory and the ability to store and manipulate oralinformation in working memory at the same time.

Assessment of patients’ voluntary attention, flexibilityof thought, information processing speed, and ability to per-form a specified task correctly, tested with the Stroop test,

The Neurodegenerative Process in Multiple Sclerosis 909

Fig. 1. MRI image of the “zone of interest” used for PMRS analysis.

demonstrated significant impairments in MS patients. Theresults from the first series of tests were not significantly dif-ferent from those of the control group – the number of wordsread without mistakes (W) was 90.46 ± 9.0 (p > 0.05). At thesame time, the results from the second series showed that thenumber of correctly named colors (C), 75.42 ± 14.0, wasalready significantly different from that in the control group(p = 0.0001). The most marked changes were obtained in thethird test series – the number of correctly read words in con-ditions of interference (CW) – was 45.6 ± 9.4, which wassignificantly different from the control group (p = 0.0001).

Thus, psychological-psychiatric testing of patientswith rMS revealed significant impairments to cognitivefunctions.

MRI Assessment of the severity of the neurodegenera-tive process in patients with rMS. Brain MRI data demon-strated foci of increased intensity on T2-WI in all patientswith rMS, the number of foci ranging from five to 50 (mean21.0 ± 12.6) foci. The number of foci on T1-WI varied from0 to 25 (mean 6.6 ± 7.0). In two patients (7.7%), initialtomograms showed no T1 foci. MRI with contrast enhance-ment did not show pathological foci in eight patients(30.8%). Several accumulation versions were performed:single, “ring” type, and “half-ring” type, and these reflect-ed different times since focus formation.

Correlation analysis did not identify any correlationsbetween disease severity on the EDSS and the number offoci on T2- and T1-WI (p > 0.05). There were also no cor-relations between the severity of cognitive impairments andthe number of foci on T2- and T1-WI (p > 0.05). There wasno relationship between neurological deficit and the pres-ence/absence or number of contrast-accumulating foci.

Thus, these studies showed that the level of neurologi-cal and cognitive deficit was not determined by the extent offocal brain damage in patients with rMS.

Studies of the control group of patients without neuro-logical disease showed that the mean NAA/Cr ratio was2.45 ± 0.19 (2.33–2.66). The mean NAA/Cr in the ZI for thegroup of patients with rMS was 2.0 ± 0.2 (1.53–2.44).Comparative analysis showed that while the age of thegroup of patients with MS was not significantly differentfrom that of the control group, the NAA/Cr ratio in patientswith MS was significantly different (p = 0.008, Mann–Whitney test) on nonparametric analysis (Fig. 2). This leadsto the conclusion that patients with MS have significantlylower NAA/Cr ratios than healthy people of the same age.

Decreases in the NAA/Cr ratio in demyelination fociwere more significant than for the ZI on average, as the latterincludes ANWM. Analysis of individual foci showed that therange of NAA/Cr ratios was 0.59–1.6, which was accompa-nied by a parallel increase in the lactate peak. However, con-sidering the fact that the increase in disability in MS is due notonly to focal changes, only deviations in the mean NAA/Crratio were monitored, these providing an overall reflection ofthe dynamics of focal and diffuse degenerative changes inbrain tissue. The selected brain zone – the corpus callosumand its adjacent white matter – allows the state of most of thelong conduction pathways of the brain to be evaluated.

Analysis of NAA/Cr ratios at one year of observationdemonstrated a significant decrease, to 1.92 ± 0.22 (p = 0.006compared with baseline), which was accompanied by anincrease in the degree of disability at one year to 2.36 ± 1.05points, which was significantly different from controls (p == 0.007).

Correlation analysis demonstrated a significant inverserelationship between disease severity and NAA/Cr in thestudy area of the brain in patients with rMS (Spearman test,r = –0.54, p = 0.006); there was also a relationship betweendisease duration and NAA/Cr (r = –0.46, p = 0.02).

Davydovskaya, Boiko, Podoprigora, et al.910

TABLE 2. Psychological and Psychiatric Testing of MS Patients and the Control Group

Note. *Significant difference from control group, p < 0.05.

Fig. 2. Significant differences between NAA/Cr in rMS patients and thecontrol group.

Correlation analysis revealed a direct and significantrelationship between performance on the Stroop test and theNAA/Cr ratio in brain tissue – the correlation coefficientwas r = 0.47 (p = 0.005) (Fig. 3). A similarly strong directrelationship was also obtained for results from the PASATtest: r = 0.44, p = 0.01. This relationship provides evidencethat the CNS content of the neuron metabolite is determinednot only by the severity of the neurological deficit, but alsoby the extent of cognitive impairments in patients with MS.

At the same time, correlation analysis using the non-parametric Spearman method showed that there were norelationships between the NAA/Cr ratio and patients’ ages,gender, age at disease onset, frequency of exacerbations, ornumbers of T2 and T1 contrast-accumulating foci (p > 0.05in all cases).

Thus, stable neurological deficit and cognitive impair-ments showed little dependence on clinical disease activityor the number of foci on T2- and T1-WI. The extents of

cognitive impairments did not correlate with focal changeson MR tomograms.

It is now recognized that the leading role in increasesin neurological and cognitive deficit in MS is played by dif-fuse degeneration processes in ANWM and ANGM in thebrain and spinal cord. Our data indicate that increases instable neurological and cognitive impairments are to a sig-nificant extent determined by changes in the NAA/Cr level,which provides an overall reflection of focal and diffusechanges in brain tissue. Patients with stable neurologicaldeficit assessed on the EDSS had more marked reductionsin the NAA/Cr ratio.

We identified a significant relationship between theextent of cognitive impairments detected with the PASATand Stroop tests and changes in the NAA/Cr ratio in the cor-pus callosum. The corpus callosum is known to play amajor role in interhemisphere interactions in the brain; thisarea carries most of the cognitive pathways [22]. Our dataare consistent with results from recent studies showing thatcognitive impairments in MS patients are to a large extentdetermined by structural changes in the white matter aroundthe corpus callosum [25].

Effects of treatment with first-line MSCMA – IFNβ-1a– on changes in the NAA/Cr ratio in MS patients. Treatmentwith IFNβ-1a given intramuscularly led to clinical stabi-lization of disease, which was demonstrated by a number ofpoints: during treatment with IFNβ-1a, none of the patientsshowed any exacerbation during the year of treatment, com-pared with a mean of 1.6 ± 0.46 exacerbations during theyear before the study (p < 0.0001). During IFNβ-1a treat-ment, patients remained clinically stable: there were noreductions in EDSS scores (1.75 ± 0.59 before treatmentand 1.75 ± 0.59 at one year of treatment). There were nochanges in cognitive test results.

Positive results were also obtained showing suppres-sion of MRI disease activity on the background of IFNβ-1atreatment: the number of foci on T2-WI showed no signifi-cant change after one year of treatment (22.1 ± 12.1 foci,p > 0.05). The number of foci on T1-WI also showed nochange after one year of treatment (4.8 ± 4.8, p > 0.05). Onthe background of treatment, only one patient showed per-sistence of contrast accumulation, though this occurred insix patients before treatment (p < 0.0001).

The initial mean NAA/Cr ratio for this group of patientswas 2.02 ± 0.16 (1.70–2.23). An important result was theobservation of the NAA/Cr ratio in the ZI showed no signif-icant change at one year of treatment and was 1.94 ± 0.14(p > 0.05, Wilcoxon test, Fig. 4).

Thus, dynamic PMRS observations demonstrated sta-bilization of the NAA/Cr ratio in brain tissue in patientswith rMS treated with i.m. IFNβ-1a for one year, while forthe overall group of rMS patients there was a significantdecrease in the NAA/Cr ratio.

During this study, we did not see any improvements inthe NAA/Cr ratio on the background of treatment, as report-

The Neurodegenerative Process in Multiple Sclerosis 911

Fig. 3. Stroop test results against NAA/Cr ratio.

Fig. 4. Dynamics of NAA/Cr ratio at one year of IFNβ-1a treatment.

ed by Khan et al. [17], which may be explained by the lim-ited nature of the observations. At the same time, our resultsare more consistent with the pathogenetic concept of thenature of nerve cell damage in rMS, which is for the greaterpart irreversible [21].

Thus, analysis of current immunomodulatory treat-ment of MS exemplified by the use of IFNβ-1a demonstrat-ed stabilization of the neuroprotective effect over one yearof treatment, which may be associated with the influencesof this agent on the neurodegeneration process in MS.

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