Axonal Loss In Normal-Appearing White Matter
In Multiple Sclerosis

Recent Pathological studies have re-emphasized that Axonal injury is present in patients with Multiple Sclerosis, the most common DeMyelinating Disease of the CNS in humans.

However, the temporal profile of DeMyelination and Axonal loss in Multiple Sclerosis patients and their independent contributions to clinical and ElectroPhysiological abnormalities are not completely understood.

In this study, we used the Theiler's murine EncephaloMyelitis Virus model of Progressive CNS Inflammatory DeMyelination to demonstrate that DeMyelination in the Spinal Cord is followed by a loss of medium to large Myelinated Fibers.

By measuring Spinal Cord areas, Motor Evoked Potentials, and Motor coordination and balance, we determined that Axonal Loss following DeMyelination was associated with ElectroPhysiological abnormalities and correlated strongly with Reduced Motor Coordination and Spinal Cord Atrophy.

These findings demonstrate that Axonal Loss can follow Primary, Immune-mediated DeMyelination in the CNS and that the severity of Axonal loss correlates almost perfectly with the degree of Spinal Cord Atrophy and Neurological Deficits.

We assessed Axonal Loss in the Normal-Appearing White Matter of the Corpus Callosum in postmortem Brains of patients with Multiple Sclerosis, using quantitative measures of both Axonal Density and White Matter Atrophy.

The calculated total number of Axons was reduced significantly (mean +/- SD, 5.4 x 10(7) +/- 3.1 x 10(7)) compared with normal controls (11.6 x 10(7) +/- 2.2 x 10(7), p = 0.001).

With a reduction both in Axonal density (median, 34%; range, 16-56%; p = 0.004) and area (mean +/- SD: Multiple Sclerosis, 584 +/- 170 mm2; controls, 871 +/- 163 mm2; p = 0.004).

These results confirm substantial Axonal loss in the Normal-Appearing White Matter and demonstrate that measures of both Axonal Density and White Matter volume are necessary to appreciate the full extent of Axonal Loss.

Damage to Axons is taken as a key factor of Disability in Multiple Sclerosis, but its PathoGenesis is largely unknown.

Axonal injury is believed to occur as a consequence of DeMyelination and was recently shown to be a feature even of the early disease stages.

The present study was aimed at characterizing the association of Axonal injury and HistoPathological hallmarks of Multiple Sclerosis such as DeMyelination, Cellular Infiltration and expression of Inflammatory Mediators.

Therefore, Axon reduction and signs of acute Axonal damage were quantified in early Lesion development of Chronic Multiple Sclerosis and correlated with DeMyelinating activity and Inflammation.

Patients with Secondary/Progressive Multiple Sclerosis revealed the most pronounced Axonal injury, whereas Primary/Progressive Multiple Sclerosis patients surprisingly showed relatively little acute Axonal injury.

Acute Axonal damage, as defined by the accumulation of Amyloid Precursor Protein (APP), was found to occur not only in active DeMyelinating but also in ReMyelinating and inactive DeMyelinated lesions with a large inter-individual variability.

Only few ReMyelinating lesions were adjacent to areas of active DeMyelination. In this minority of lesions, Axonal damage may have originated from the neighborhood.

APP expression in damaged Axons correlated with the number of Macrophages and CD8⁺ T-Lymphocytes within the lesions.

But not with the expression of Tumor Necrosis Factor-alpha (TNF-) or inducible Nitric Oxide Synthase (iNOS).

Axonal injury is therefore, at least in part, independent of DeMyelinating activity, and its PathoGenesis may be different from DeMyelination.

This has major implications for therapeutic strategies, which aim at preventing both DeMyelination and Axonal Loss.

The idea that the initiating event in the formation of all new Multiple Sclerosis lesions is a focal Blood-Brain Barrier (BBB) leakage associated with PeriVascular Inflammation has been challenged recently.

By the observation of subtle abnormalities in some quantitative Magnetic Resonance (MR) parameters (including the Magnetization Transfer Ratio) prior to lesion enhancement.

MR Diffusion imaging can non-invasively quantify the average Apparent Diffusion Coefficient (av ADC).

A measure of water molecule random motion that is sensitive to pathological change in Multiple Sclerosis lesions and to abnormalities in the Normal-Appearing White Matter (NAWM).

We therefore used MR Diffusion imaging to investigate the dynamic evolution of water Diffusion measurements in new enhancing Multiple Sclerosis Lesions, in the NAWM from which they arise, and in anatomically matched ContraLateral NAWM regions from which no visible lesions develop.

Gadolinium Diethylenetriaminepentaacetic Acid (Gd)-enhanced MRI and MR Diffusion studies were performed monthly for 1 year in five Multiple Sclerosis patients with clinically and radiologically active disease.

The ADCav was calculated at each time point of the study (before, during and after lesion appearance on Gd-enhanced scans) for each new enhancing lesion, and for regions matched for size and position in the ContraLateral NAWM.

A steady and moderate increase in ADCav in PreLesion NAWM was observed, which was followed by a rapid and marked increase at the time of Gd enhancement and a slower decay after the cessation of enhancement.

In matched ContraLateral NAWM regions there was a significant but milder increase in ADCav at the time of the first noted lesion enhancement.

These findings indicate that New Focal Lesions associated with frank BBB leakage are preceded by subtle, progressive alterations in tissue integrity beyond the resolution of conventional MRI.

The increases in ADCav in anatomically matched ContraLateral regions after lesions have appeared supports the concept that Structural Damage in lesions causes damage or dysfunction in connected areas of NAWM.

Recent observations made using Magnetic Resonance Spectroscopy to measure the Axonal marker N-AcetylAspartate have emphasized the fact that substantial Axonal damage occurs in Multiple Sclerosis, in addition to DeMyelination.

The Axonal damage is present both in Lesions and Normal-Appearing White Matter, progresses over time, and correlates with clinical disability.

These observations, together with observations demonstrating that adaptations of Sodium channels can restore Conduction in DeMyelinated Axons, have led to the hypothesis that Axonal Damage may be responsible for a significant proportion of the chronic disability that accrues in MS.

Brain Atrophy measurement can provide an estimate of the amount of tissue destruction due to the pathologic processes in Multiple Sclerosis.

The potential usefulness of Atrophy as a marker of disease progression depends upon the concurrent and predictive relationships between Atrophy and Disability.

A follow-up study was performed to measure Atrophy and Disability Scores in patients from the Multiple Sclerosis Collaborative Research Group's Phase III trial of IFN-ß-1a (Avonex) in Relapsing/Remitting Multiple Sclerosis.

New data were obtained on 160 out of 172 eligible patients from the original trial were enrolled in the follow-up study approximately 8 years after randomization.

The follow-up visit consisted of several tests and questionnaires including a clinical exam to determine Expanded Disability Status Score (EDSS) and Multiple Sclerosis Functional Composite (MSFC), and a MRI exam to calculate the Brain Parenchymal Fraction.

Brain Parenchymal Fraction was correlated with both EDSS and MSFC at each of the four time points for which data were available (baseline 1, 2 and 8 years).

Furthermore, the change in BPF was correlated with the changes in Disability Scores from the end of the Phase III trial to the follow-up exam.

These data suggest that Brain Atrophy may be a useful and clinically relevant marker of disease progression in Relapsing/Remitting MS.

Objective
Brain Atrophy may occur early in the course of Multiple Sclerosis (MS) and may be associated with disability.

Brain Magnetic Resonance Imaging (MRI) of 114 MS patients (group A) were analyzed for Regional Atrophy (vs age-/gender-matched controls) and T₁ and T₂ lesions using 4-point rating systems.

Thirty-five separate patients (group B) were analyzed for Cortical Atrophy (ordinal scale), Third Ventricular Width, and total T₂ HyperIntense Lesion Volume (computer assisted).

In group A, regression modeling indicated that Inferior Frontal Atrophy (P = .0003) and T₂ lesions in the Pons (P = .02) predicted physical disability (Expanded Disability Status Scale [EDSS] score).

Secondary/Progressive (S/P) versus Relapsing patients were predicted by Inferior Parietal (P = .002), Superior Parietal (P = .006), Temporal (P = .008), Inferior Frontal (P = .01), Superior Frontal (P = .01), Cerebellum (P = .01), Occipital (P = .01), and MidBrain (P = .02) Atrophy.

S/P patients were also predicted by total Atrophy (P = .01) and Third Ventricular enlargement (P = .03) but not T₁ or T₂ lesions.

In group B, the regression model predicting EDSS score included only Superior Frontal Atrophy (r = 0.515, P = .002).

Mean kappa coefficients of ordinal ratings were 0.9 (intraobserver) and 0.8 (interobserver). Ordinal ratings correlated well with quantitative assessments.

The authors conclude that Brain Atrophy is closely associated with physical disability and clinical course in MS patients and can be appreciated using a semiquantitative MRI regional rating system.

Background
Brain imaging studies detect abnormalities in Normal-Appearing White Matter in patients with MS.

Objective
To investigate the HistoPathologic basis for these changes in autopsy tissue from a patient with MS with 9 months' disease duration and a terminal BrainStem lesion.

Methods
The BrainStem and Spinal Cord were analyzed UltraStructurally and ImmunoCytoChemically for Axons, Myelin, and Activated Microglia/Macrophages.

Results
Pathologic findings were consistent with a terminal inflammatory DeMyelinated Lesion at the CervicoMedullary junction.

The Ventral Spinal Cord Column, containing Descending Tracts, exhibited 22% Axonal loss at segment C7, but grossly normal ImmunoStaining for Myelin.

Confocal and electron microscopy revealed Myelin Sheaths without Axonal content and initial stages of Myelin degradation by activated Microglia/Macrophages among intact Myelinated Axons.

Axonal number and appearance was normal in Ascending Sensory Tracts.

Conclusions
These studies confirm Axonal Degeneration in the absence of Myelin loss as one HistoPathologic correlate to abnormal MR findings in patients with MS.

Axonal damage in Multiple Sclerosis has become an important issue. This has been emphasized by recent in vivo proton Magnetic Resonance Spectroscopy (MRS) and in vitro pathology studies that have found Axonal damage in both lesions and the surrounding Normal-Appearing White Matter.

In particular, proton MR Spectroscopy, by monitoring levels of N-AcetylAspartate (a putative marker of Axonal integrity), has been particularly illuminating.

As the extent of Axonal injury associated with White Matter Inflammation and DeMyelination had not been well appreciated from classical pathology studies.

Recent MR data demonstrate that Cerebral Axonal damage begins and contributes to disability from the earliest stages of the disease.

This implies that the apparently primary role of Axonal damage and loss in the pathogenesis of the disease should be given due importance, and argues for the early treatment of Multiple Sclerosis with agents directed not only against inflammation, but also towards Axonal protection.

The traditional notion that Multiple Sclerosis is a primary DeMyelinating Disease has led to a plaque-centred view of both aetiology and the pathogenesis of disease progression. The presence of Axonal Loss has received increasing recognition.

However, the relative roles of DeMyelination and Axonal Loss have not been fully clarified in Multiple Sclerosis nor have their possible interrelationships been elucidated.

Post-mortem material from the Cerebrum, BrainStem and Spinal Cord of 55 Multiple Sclerosis patients (29 males) with an age range of 25-83 years (mean = 57.5 years) and length of disease history ranging from 2 to 43 years (mean = 17.1 years) was stained for Myelin.

Plaque load was calculated by summing the relative proportion of plaque area compared with total White Matter area of the CorticoSpinal and Sensory Tracts at each level.

This was related to estimates of Axonal density and of total Axon number in these tracts in the Spinal Cord. Our results indicate that plaque load did not correlate with Brain weight.

Unexpectedly, after adjusting for sex, age and duration of disease, correlations between total plaque load and Axonal Loss in both the CorticoSpinal Tract and Sensory Tracts were weak or absent at each level investigated.

Since there was little correlation between plaque load and Axonal Loss, the possibility that DeMyelination is not the primary determinant of Spinal Cord Axonal Loss warrants consideration.

Objectives
Recent studies have indicated that Brain Atrophy is more closely associated with Cognitive Impairment in Multiple Sclerosis (MS) than are conventional MRI lesion measures.

Enlargement of the Third Ventricle shows a particularly strong correlation with Cognitive Impairment, suggesting clinical relevance of damage to surrounding structures, such as the Thalamus.

Previous imaging and pathology studies have demonstrated Thalamic involvement in MS.

In this study, we tested the hypothesis that Thalamic Volume is lower in MS than in normal subjects, and that Thalamic Atrophy in MS correlates with Cognitive function.

Methods
We studied 79 patients with MS and 16 normal subjects. A subgroup of 31 MS subjects underwent Cognitive testing.

The Thalamus was segmented in whole from three-dimensional MRI scans.

We also determined Whole Brain Atrophy (Brain Parenchymal Fraction), Third Ventricular Width, and Whole Brain T₂-weighted (Fluid-Attenuated Inversion Recovery) HyperIntense, T₁ HypoIntense, and Gadolinium-enhanced lesion volumes.

Results
Normalized Thalamic Volume was 16.8% lower in the MS group (p < 0.0001) vs controls.

Cognitive performance in all domains was moderately to strongly related to Thalamic Volume in the MS group (r = 0.506 to 0.724, p < 0.005).

And, Thalamic Volume entered and remained in all regression models predicting Cognitive performance. Thalamic Volume showed a weak relationship to physical disability score (r = -0.316, p = 0.005).

Conclusion
These findings suggest that Thalamic Atrophy is a clinically relevant biomarker of the NeuroDegenerative Disease process in Multiple Sclerosis.