Axons & MS Disability

In this review, data is summarized supporting the hypothesis that Axonal loss is a major pathologic process responsible for irreversible Neurologic Disability in patients with Multiple Sclerosis.

Pathologic studies implicate Inflammatory DeMyelination as a principal cause of Axonal transection and subsequent Axonal degeneration.

Axonal degeneration (Wallerian Degeneration) caused by Chronic DeMyelination in the absence of active inflammation may also contribute to Progressive Disability in the later stages of the disease.

Studies using Magnetic Resonance Spectroscopy suggest that Axonal Loss begins at the onset of the disease, and studies using Magnetic Resonance Imaging have documented Brain Atrophy in the earliest stages of Multiple Sclerosis.

Brain Atrophy increases during the Relapsing/Remitting Disease stage without concurrent disability progression.

This suggests that compensatory mechanisms maintain Neurologic function, despite progressive Brain tissue loss during the early stages of the Disease.

Beyond a threshold, however, further Axonal loss leads to continuously Progressive Neurologic Disability.

We hypothesize that the rate and extent of Axonal Loss during Relapsing/Remitting Multiple Sclerosis determines, when a patient enters the Secondary/Progressive stage of the Disease.

This view of disease PathoGenesis has several important implications:

• Surrogate markers of Axonal loss are needed to monitor the disease process for patient care and for clinical trials.
  • We propose Brain Parenchymal Fraction, a precise measure of Whole-Brain Atrophy, as an attractive candidate for this purpose.

• Disease-modifying therapy should be used early in Multiple Sclerosis patients, before extensive Axonal Loss has occurred.

• NeuroProtective drugs should be tested in combination with AntiInflammatory drugs in Multiple Sclerosis patients.

• Finally, studies of the time course of Axonal Loss, and its mechanisms are critical for effective therapeutic intervention.

Background
Multiple Sclerosis is an inflammatory DeMyelinating Disease of the Central Nervous System and is the most common cause of Neurologic disability in young adults.

Despite AntiInflammatory or ImmunoSuppressive therapy, most patients have Progressive Neurologic deterioration that may reflect Axonal loss.

We conducted pathological studies of Brain tissues to define the changes in Axons in patients with Multiple Sclerosis.

Methods
Brain tissue was obtained at autopsy from 11 patients with Multiple Sclerosis and 4 subjects without Brain disease.

Fourteen active Multiple-Sclerosis lesions, 33 Chronic active Lesions, and samples of Normal-Appearing White Matter were examined for DeMyelination, Inflammation, and Axonal pathologic changes by ImmunoHistoChemistry and confocal microscopy.

Axonal transection, identified by the presence of terminal Axonal ovoids, was detected in all 47 Lesions and quantified in 18 Lesions.

Results
Transected Axons were a consistent feature of the lesions of Multiple Sclerosis, and their frequency was related to the degree of Inflammation within the lesion.

The number of Brain transected Axons per cubic millimeter of tissue averaged:

We used computer pattern recognition of Proton Magnetic Resonance Spectroscopic image data to differentiate between Brain Tumors and large, isolated, DeMyelinating lesions of the type seen in Multiple Sclerosis.

Leave-one-out linear discriminant analyzes correctly classified resonance profiles from five acute DeMyelinating lesions, 20 low-grade Astrocytomas, 22 Anoplastic Astrocytomas, and 24 Glioblastomas.

Classification of nonacute Lesions will require further development, as the metabolic profiles of DeMyelinating lesions evolve over time.

The current study was designed to determine the relative distribution of decreases of N-AcetylAsparate (NAA), a marker of Axonal damage, between lesions and Normal-Appearing White Matter (NAWM) of patients with established Multiple Sclerosis.

And to test for associations between changes in the ratio of NAA to Creatine/PhosphoCreatine (NAA:Cr) in those compartments and changes in disability.

Data were collected from a 30-month longitudinal study of 28 patients with either a Relapsing course with partial remissons and no progression between attacks (Relapsing/Remitting) (11 patients).

Or a course of progressively increasing disability, following a period of Relapsing disease (Secondary/Progressive) (17 patients).

Proton Magnetic Resonance Spectroscopic Imaging (MRSI) and conventional MRI examinations were performed at 6-8-month intervals with concurrent clinical assessments of disability.

General linear models were used to test associations between MRSI, MRI, Lesion volume and clinical data.

Analysis confirmed the NAA:Cr ratio is lower in lesions than in NAWM:
• -15.3% in Relapsing/Remitting Multiple Sclerosis
• -8.8% in Secondary/Progressive Multiple Sclerosis

The lower NAA:Cr ratio per unit lesion volume previously observed for Secondary/Progressive relative to Relapsing patients was found to result from a lower ratio (8.2%, P < 0.01) in the NAWM rather than from any differences within lesions.

The importance of changes in the NAWM was emphasized further with the observation that the NAA:Cr ratio in the Normal-Appearing White Matter:

• Accounted for most of the observed 15.6% (P < 0.001) decrease in the NAA:Cr ratio in the Brains of Relapsing/Remitting patients over the period of study.

The decrease in the NAA:Cr ratio in NAWM correlated strongly (P < 0.001) with changes in Disability in the Relapsing/Remitting subgroup.

These results add to data suggesting that Axonal damage or loss may be responsible for functional impairments in Multiple Sclerosis.

The accumulation of secondary Axonal damage in the NAWM may be of particular significance for understanding chronic disability in this disease.

The advent of Magnetic Resonance Imaging (MRI) has revolutionized the clinical approach to the evaluation of Brain White Matter Disorders and has contributed significantly to expansion of the concept of these diseases.

MRI is very sensitive at detecting White Matter lesions, but conventional T₁ and T₂-weighted images do not provide specific pathological information about the Lesions.

And correlation between MRI Lesion Load and clinical Disability is often weak.

Proton Magnetic Resonance Spectroscopy can provide chemical-pathological information of a given tissue in vivo.

The use of this MR technique in Brain White Matter Disorders has been shown to improve diagnostic classification and to provide surrogate measures useful for monitoring disease evolution and response to therapeutic intervention.

In a 6-year longitudinal study of a patient with Progressive/Relapsing Multiple Sclerosis (MS), we used Proton Magnetic Resonance Spectroscopy to assess N-AcetylAspartate (NAA).

From a large central Brain Volume to evaluate the relationship between this marker of Neuronal integrity and clinical disability.

During the follow-up period, there was one major relapse and its subsequent partial remission.

Changes in the Brain NAA to Creatine ratio correlated strongly with clinical disability (Spearman rank coefficient = -0.73, p < 0.001). We interpret this as evidence that Axonal dysfunction or loss contributes to functional impairment of patients with MS.

Because the NAA signal in the large volume of interest originated predominantly from White Matter that appeared normal on conventional MRI, these results also suggest that some degree of Axonal dysfunction may be widespread in acute, severe relapses.

Oligodendrocyte and Axon pathology was studied in 11 autopsy cases of clinically silent Multiple Sclerosis.

A total of 54 lesions, either DeMyelinated or late ReMyelinated, were distributed through the whole Brain and Spinal Cord with 39% of the lesions located in PeriVentricular areas.

Determination of Axon density revealed an average reduction of 64% and 59% in DeMyelinated and ReMyelinated Lesions with an extreme variation between different plaques and cases.

Oligodendrocytes were identified by ImmunoCytoChemistry for Myelin Oligodendrocyte Glycoprotein (MOG) and in situ hybridization for ProteoLipid Protein (PLP) mRNA.

Oligodendrocytes were almost completely lost in DeMyelinated lesions; ReMyelinated lesions revealed preservation of a considerable number of Oligodendrocytes within the lesions.

At the border between plaques and the PeriPlaque White Matter, similar Oligodendrocyte numbers as in ReMyelinated lesions were found.

Different factors including Lesion site, Axonal preservation and ReMyelination may thus contribute to the Clinical NonAppearance of Multiple Sclerosis Lesions.

One of the histological hallmarks of early Multiple Sclerosis lesions is primary DeMyelination, with Myelin destruction and relative sparing of Axons.

On the other hand, it is widely accepted that Axonal loss occurs in, and is responsible for, the permanent Disability characterizing the later Chronic Progressive stage of the disease.

In this study, we have used an AntiBody against Amyloid Precursor Protein, known to be a sensitive marker of Axonal Damage in a number of other contexts, in ImmunoCytoChemical experiments on paraffin embedded Multiple Sclerosis Lesions of varying ages.

In order to see at which stage of the disease Axonal damage, in addition to DeMyelination, occurs and may thus contribute to the development of disability in patients.

The results show the expression of Amyloid Precursor Protein in damaged Axons within acute Multiple Sclerosis lesions, and in the active borders of less acute Lesions.

This observation may have implications for the design and timing of therapeutic intervention, one of the most important aims of which must be the reduction of permanent Disability.

Axonal pathology is a major cause of Neurological disability in Multiple Sclerosis. Axonal transection begins at disease onset but remains clinically silent because of compensatory Brain mechanisms.

Noninvasive surrogate markers for Axonal Injury are therefore essential to monitor cumulative disease burden in vivo.

The Neuronal compound N-AcetylAspartate, as measured by Magnetic Resonance Spectroscopy, is currently the best and most specific noninvasive marker of Axonal pathology in Multiple Sclerosis.

The possibility has been raised, however, that N-AcetylAspartate is expressed also by OligodendroGlial Lineage Cells. In order to investigate N-AcetylAspartate specificity for White Matter Axons, transected rat Optic Nerves were analyzed by high-performance liquid Chromatography and ImmunoHistoChemistry.

In transected adult Nerves, N-AcetylAspartate and N-Acetyl AspartylGlutamate decreased in concordance with Axonal Degeneration and were undetectable 24 days PostTransection.

Nonproliferating Oligodendrocyte Progenitor Cells, Oligodendrocytes, and Myelin were abundant in these Axon-free Nerves. At 24 days PostTransection, N-AcetylAspartate was increased (42%; p = 0.02) in nontransected ContraLateral Nerves.

After transection at postnatal day 4, total N-AcetylAspartate decreased by 80% (P14; p = 0.002) and 94% (P20; p = 0.003). In these developing Axon-free Nerves, 25 to 33% of Oligodendrocyte Progenitor Cells were proliferating.

These data validate Magnetic Resonance Spectroscopy measurements of N-AcetylAspartate as an Axon-specific monitor of Central Nervous System White Matter in vivo.

In addition, the results indicate that Neuronal adaptation can increase N-AcetylAspartate levels, and that 5 to 20% of the N-AcetylAspartate in developing White Matter is synthesized by proliferating Oligodendrocyte Progenitor Cells.

Objective
The cause of Axonal Loss, an important contributor to disability in MS, is poorly understood.

This study investigated whether progression in MS is associated with CSF AntiBodies to the 68-kd light NeuroFilament subunit (NF-L), an Axonal Cytoskeletal protein, and compared this with AntiBodies against Tubulin and the heavy NeuroFilament subunit (NF-H).

Methods
IgG to NF-L, Tubulin, and NF-H was measured by ImmunoAssay in matched CSF and Serum samples from patients with Relapsing/Remitting MS (RRMS; n = 39), Primary/Progressive MS (PPMS; n = 10), and Secondary/Progressive MS (SPMS; n = 18); patients with Other Inflammatory (n = 21) and NonInflammatory (n = 40) Neurologic Diseases; and healthy controls (n = 12).

ImmunoCytoChemistry was performed to assess AntiBody binding to human Brain sections, and IsoElectric Focusing with ImmunoBlotting was performed to assess OligoClonal Anti-NF-L production.

Results
Intrathecal production of Anti-NF-L AntiBodies was significantly elevated in PPMS and SPMS.

In contrast, there were no significant differences in CSF levels of AntiBodies to Tubulin or NF-H between the groups.

Anti-NF-L, AntiTubulin, and anti-NF-H levels correlated with the duration of disease before Lumbar Puncture and Expanded Disability Status Scale levels.

ImmunoCytoChemistry demonstrated binding of CSF or Serum AntiBodies to Axonal or Neuronal components in six of seven RRMS patients, seven of seven PPMS patients, and eight of 10 SPMS patients tested.

IsoElectric Focusing demonstrated independent CSF OligoClonal Bands reactive with NF-L in six of 13 specimens tested.

Conclusion
Anti-NF-L AntiBodies seem to be raised in Progressive MS and may serve as a marker for Axonal Loss and disease progression. They may contribute to Axonal Loss and the accumulation of disability.

There is still a controversy regarding the best Regional Brain Atrophy measurements in Multiple Sclerosis (MS) studies.

The aim of this study was to establish whether, in a Cross-Sectional Study, the Normalized measurements of Regional Brain Atrophy correlate better with the MRI-defined regional Brain lesions than the Absolute measurements of Regional Brain Atrophy.

We assessed 45 patients with Clinically Definite Relapsing/Remitting (RR) MS (median disease duration 12 years), and measured T₁-lesion load (LL) and T₂-LL of Frontal Lobes and Pons, using a reproducible semi-automated technique.

The Regional Brain Parenchymal Volume (RBPV) of Frontal Lobes and Pons was obtained by use of a computerized interactive program, which incorporates semi-automated and automated segmentation processes.

A normalized measurement, the Regional Brain Parenchymal Fraction (RBPF), was calculated as the ratio of RBPV to the total Volume of the Parenchyma and the CerebroSpinal Fluid (CSF) in the Frontal Lobes and in the region of the Pons.

The Total Regional Brain Volume Fraction (TRBVF) was obtained after we had corrected for the Total Volume of the Parenchyma and the CSF in the Frontal Lobes and in the region of the Pons for the Total IntraCranial Volume.

The mean Coefficient of Variation (CV) for RBPF of the Pons was 1% for intra-observer reproducibility and 1.4% for inter-observer reproducibility.

Generally, the Normalized measurements of Regional Brain Atrophy correlated with Regional Brain Volumes and disability better than did the Absolute measurements.

RBPF and TRBVF correlated with T₂-LL of the Pons (r=-0.37, P=0.011, and r= -0.40, P=0.0005 respectively) and with T₁-LL of the Pons (r=-0.27, P=0.046, and r=-0.31, P=0.04, respectively).

Whereas RBPV did not (r=-0.18, P = NS). T₁-LL of the Frontal Lobes was related to RBPF (r=-0.32, P=0.033) and TRBVF (r=-0.29, P=0.05), but not to RBPV (R=-0.27, P= NS).

There was only a trend of correlation between T₂-LL of the Frontal Lobes and RBPF (r=-0.27, P=0.06) and TRBVF (r=-0.28, P=0.057), and no correlation with RBPV (r=-0.23, P= NS).

The magnitude of correlation between the Expanded Disability Status Scale (EDSS) and Pontine and Frontal Lobe RBPF and TRBVF was more than twice as high as the correlation between EDSS and RBPV of the same regions.