Axonal Pathology In MS

The primary pathological mechanisms in Stroke and Multiple Sclerosis (MS) are very different but in both diseases, Impairment may arise from a final common pathway of Axonal Damage

We aimed to examine the relationship between Motor Impairment, Magnetization Transfer Ratio (MTR) (an index of DeMyelination), and N-AcetylAspartate (NAA) loss (an index of Axonal injury) localized to the Descending Descending Motor Pathways in Stroke and MS.

Twelve patients between 1 and 10 months after first Ischaemic Stroke causing a motor deficit and 12 patients with stable MS with asymmetric Motor Deficit were examined.

T₂-weighted imaging of the Brain together with MTR and Proton (Voxel [volume element] 1.5x2x2 cm(3)) MRS localized to the Posterior Limb of the Internal Capsule were performed and correlated to a composite Motor Deficit score.

MTR and NAA in the Internal Capsule were reduced in both Stroke and MS patients compared to controls. NAA loss correlated with Motor Deficit score in both Stroke and MS (p<0.001 and p = 0.04, respectively).

Correlations were seen between MTR and Motor Deficit (p<0.001) MTR and NAA loss (p <0.001) in Stroke patients but not in MS patients.

Axonal injury in the Descending Motor Tracts would appear to be an important determinant of Motor Impairment in both Stroke and MS.

In Stroke, MTR measures of DeMyelination are closely related to Axonal damage and thus also correlate with Motor Deficit.

However in MS, MTR measures of DeMyelination do not correlate with NAA loss or Motor Deficit.

Suggesting that DeMyelination and Gliosis may occur independently of Axonal Damage and are less closely linked with Functional Impairment.

The objective of this study was to characterize and compare the Proton Magnetic Resonance Spectrum of a Voxel, containing lesions or Normal-Appearing White Matter (NAWM), in Primary/Progressive (P/P) and Secondary/Progressive (S/P) Multiple Sclerosis (MS) patients.

Single-Voxel Proton Magnetic Resonance Spectroscopy and Magnetic Resonance Imaging were performed in 35 MS patients (17 P/P and 18 S/P) and 17 controls.

Spectra from an 8 ml Voxel located in the Parieto-Occipital region were obtained with a Spin-Echo pulse sequence (1600 ms/135 ms/256, TR/TE/acquisitions).

Resonance areas due to N-AcetylAspartate (NAA), Creatine/PhosphoCreatine (Cr) and Choline compounds (Cho) were determined, and results expressed in terms of area in arbitrary units or as metabolite ratios.

With respect to the control group, there were significant reductions in NAA and NAA/Cho ratio in the P/P-lesion, S/P-lesion, P/P-NAWM and S/P-NAWM groups.

There were no significant differences between the P/P-NAWM and S/P-NAWM groups. These results support the existence of metabolic changes in the White Matter of P/P and S/P patients and suggest that there is Neuronal damage and/or loss in both clinical courses.

Finally, characterization of the Parieto-Occipital region showed no significant differences in the Spectral pattern of NAWM between P/P and S/P clinical courses of MS.

Copyright 2000 John Wiley & Sons, Ltd.

Imaging and PathoMorphological studies in Multiple Sclerosis suggest that Axonal injury and Axonal Loss are playing a crucial role in those with persistent disability and long-standing disease.

Although the existence of Axonal injury in Multiple Sclerosis is proven, especially in the zone of active inflammation, the effect of chronic inflammation on the Axons remains elusive.

The aim of this study was to perform a quantitative morphometrical analysis, estimating Axonal loss and evaluating Axonal degenerative changes in Cervical Spinal Cord samples of patients suffering from Secondary/Progressive Multiple Sclerosis.

Completely DeMyelinated plaques, Normal-Appearing White Matter (NAWM) and control material from anatomically identical regions of the Cord have been compared. NeuroFilament ImmunoStaining was used for identification of the Axons.

We observed a significant reduction of Axonal density (number of Axons/mm(2)) in Multiple Sclerosis, both in the Plaque and in the NAWM compared with the control cases.

Axons under approximately 3.3 microm diameter seemed to be more affected. The intensity of the ImmunoStaining was significantly reduced in the plaque compared with either NAWM or control.

Our results on the Cervical Cord combined with other observations support the concept of slow Axonal degeneration rather than acute damage as a cause of chronic disability in Multiple Sclerosis.

• Comment in: Brain 2000 Feb;123 ( Pt 2):203-4

To test for Axonal damage or dysfunction in White Matter Tracts remote from acute DeMyelinating lesions in the White Matter of the Normal-Appearing Hemisphere of three patients with large, solitary Brain DeMyelinating Lesions of the type seen early in Multiple Sclerosis.

We used Brain Proton Magnetic Resonance Spectroscopic imaging to measure changes in N-AcetylAspartate (NAA), an index of Neuronal integrity.

During the acute phase of their disease, all patients showed normal ratios of NAA to Creatine (Cr) resonance intensity throughout the Hemisphere ContraLateral to the lesion.

However, on examination 1 month later, all of the patients showed abnormally low NAA/Cr resonance intensity ratios (reduction of NAA/Cr by 22-35%) in Voxels of the ContraLateral (opposite side) Hemisphere which were homologous to the DeMyelinating lesion.

Other Voxels in the Normal-Appearing Hemisphere showed normal NAA relative resonance intensities.

The decrease in NAA/Cr in Voxels of the Normal-Appearing Hemispheres resolved in all patients after 6 months, with a time course similar to that observed for NAA from Voxels within the Lesions.

We conclude that effects of damage or dysfunction to Axons traversing inflammatory lesions can be transmitted over long distances in the NAWM.

Such remote, secondary effects may be an expression of dysfunction of Axons (Wallerian Degeneration) in Projection Pathways or of the reorganization of Functional Pathways seen in Brains recovering from an acute injury.

HypoIntense T₁ lesions in Multiple Sclerosis patients correlate with Axonal loss at autopsy and biopsy.

We evaluated the chemical substrate of HypoIntense T₁ lesions by using in vivo Proton Magnetic Resonance Spectroscopy, and analyzed the Spectroscopic correlate of increased T₁-relaxation time measurements.

Localized Proton Magnetic Resonance Spectroscopy and T₁-relaxation time measurements were performed in Lesions.

Selected on T₁-weighted Spin-Echo Magnetic Resonance Images according to degree of HypoIntensity, in Normal-Appearing White Matter (NAWM) and in normal White Matter of controls.

In NAWM, prolongation of T₁-relaxation time and a decrease in N-AcetylAspartate (NAA) were present, compared with normal White Matter.

Severely HypoIntense lesions showed a lower concentration of NAA and Creatine compared with NAWM and a lower concentration of NAA compared with IsoIntense to mildly HypoIntense lesions.

NAA concentration correlated with degree of HypoIntensity of lesions and with T₁-relaxation time within the Spectroscopic Voxel.

Our results provide the first in vivo evidence of Axonal Damage in severely HypoIntense T₁ lesions in Multiple Sclerosis patients.

T₁-relaxation time correlates with the concentration of NAA in both Multiple Sclerosis Lesions and NAWM, indicating that this parameter deserves further evaluation to monitor disease progression.

The purpose of this study was to compare Magnetic Resonance Imaging (MRI) features and Proton MR Spectroscopy (1H-MRS) patterns of Multiple Sclerosis (MS) plaques in order to define the metabolic substrate in different lesion subtypes.

Combined MRI and single-Voxel 1H-MRS investigation was performed in 54 MS patients:

1. 47 Relapsing/Remitting (RR)
2. 07 Secondary/Progressive (SP)

From sixty-seven Gadolinium (Gd) enhancing MS lesions, on pre-contrast T₁-weighted scans:

1. Thirty-seven lesions were
   • 09 IsoIntense
   • 28 HypoIntense
2. On thirty Gd unenhancing T₁-weighted scans, pre- and post-contrast:
   • 06 IsoIntense
   • 24 HypoIntense

Choline (Cho), Creatine (Cr), N-AcetylAspartate (NAA) and Lactate were evaluated in 1H Spectra acquired from MS plaques and from Normal White Matter (NWM) of 22 Neurological controls.

MS lesions of RR patients were characterized by a significant increase of Cho/Cr and decrease of NAA/Cr and NAA/Cho ratios.

No significant metabolite changes were found in lesions of SP patients. Gd enhancing plaques showed Lactate signal with higher frequency (37.8%) than Gd unenhancing plaques (16.7%) (p=0.04).

A significant increase of Cho/Cr was found in Gd enhancing Lesions when compared to controls (p<0.01), and to Gd unenhancing lesions (p<0.05).

In particular, there was evidence of a significant increase of Cho/Cr in pre-contrast T₁ HypoIntense Gd enhancing lesions (p<0.01 vs. controls).

The Gd unenhancing lesions (p<0.01), in particular the T₁ HypoIntense group (p<0.05), showed a significant decrease of NAA/Cr only when compared to controls.

These data confirm that in vivo MRS indicates key pathological features of MS plaques. The increased Cho/Cr ratio found in Gd-enhancing plaques, in particular in the T₁ HypoIntense lesions, may reflect increased membrane cell turnover.

The T₁ HypoIntense Gd unenhancing Plaques better reflect Axonal Damage, as suggested by the decrease of NAA/Cr.

Nevertheless, the lack of statistical differences in NAA/Cr between plaque subgroups suggests that Axonal impairment might occur even in the early stages.

The importance of Axonal damage in Multiple Sclerosis (MS) has been recently stressed in Proton Magnetic Resonance Spectroscopy and Pathological studies, but the exact mechanism producing this damage is unknown.

The aim of our study was to ascertain whether soluble mediators present in the CerebroSpinal Fluid (CSF) of patients with Relapsing/Remitting MS could induce Neuron injury in culture.

Different biochemical and CytoChemical parameters were determined in Primary embryonal rat Neuron cultures following 8 days of exposure to CSF. CytoToxic activity was evaluated with a blue formazan production colorimetric assay.

Morphological and ImmunoCytoChemical studies performed with AntiBodies against beta-Tubulin revealed Neuritic fragmentation, Axonal damage and cellular shrinkage indicating Apoptosis.

Detection of Apoptosis was carried out using the fluorescent DNA-binding dye Hoechst 33342, as well as by a Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End-Labeling assay.

We observed that soluble factors in CSF from patients with "aggressive" MS i.e, those with poor recovery after relapses, induced Neurite breakdown and Neuronal Apoptosis in cultures. Neuron injury is not related with Blood-Brain Barrier dysfunction nor with Ig G index.

Interestingly, CSF from patients with "non-aggressive" MS i.e., Relapsing/Remitting patients with a good recovery after relapses, did not induce any damage.

In conclusion, we report that CSF from patients with aggressive MS bears soluble mediators that induce Axonal damage and Apoptosis of Neurons in culture.

These mediators can be present during the first attack of the disease, and the Neuronal Damage caused could be related to the functional deficit of these MS patients.

Objective
To assess Axonal damage and its contribution to disability at different stages of Multiple Sclerosis (MS).

Background
Recent in vivo imaging and in situ pathologic studies have demonstrated that substantial Axonal damage accompanies the inflammatory lesions of MS.

However, the relation of Axonal Damage to the duration of MS and its contribution to disability at different stages of the disease remain poorly defined.

Design
We performed Proton Magnetic Resonance Spectroscopic Imaging in 88 patients with a wide range of clinical disability and disease duration.

To measure N-AcetylAspartate (NAA), an index of Axonal integrity, relative to Creatine (Cr) in a large Central Brain Volume that included mostly Normal-Appearing White Matter on Magnetic Resonance Imaging.

Results
We observed that the NAA/Cr values were abnormally low in the early stages of MS, even before significant disability (measured using the Expanded Disability Status Scale [EDSS]) was evident clinically, and declined more rapidly with respect to EDSS at lower than at higher EDSS scores (P<.001).

The correlation of NAA/Cr values with EDSS score was significantly (P<.03) stronger in patients with mild Disability (EDSS score <5, Spearman rank order correlation = -0.54, P<.001) than in patients with more severe Disability (EDSSscore >/=5, Spearman rank order correlation = -0.1, P<.9).

When similar analyzes were performed in patients with MS grouped for duration of disease, the subgroup with early disease duration (<5 years) also showed central Brain NAA/Cr resonance intensity ratios significantly lower than healthy controls (P<.001).

Conclusion
Cerebral Axonal damage begins and contributes to disability from the earliest stages of the disease.

Components of the PlasMinogen Activator (PA) and Matrix MetalloProtease (MMP) cascade have been characterized in Multiple Sclerosis lesions.

By ImmunoHistoChemistry, Enzyme-linked ImmunoSorbent assay and Enzyme activity assays in order to establish a functional role for the Enzyme sequence in Lesion development.

Highly significant quantitative increases in Urokinase PA (uPA), Urokinase Receptor (uPAR) and PlasMinogen Activator Inhibitor-1 were detected.

In acute Multiple Sclerosis lesions (P < 0.0001) and in uPAR in Normal-Appearing White Matter (P < 0.0001) compared with control tissue.

All three proteins were Immunolocalized to MonoNuclear Cells in PeriVascular Cuffs and to Macrophages in the Lesion Parenchyma.

MMP-9 and the Tissue Inhibitor of MetalloProteinase-1 also increased during lesion development but the Enzyme was present largely in the inactive pro-form.

In contrast to uPA, the concentration and activity of tissue PA (tPA), the most abundant PlasMinogen Activator in normal control Brain, were reduced in Multiple Sclerosis specimens.

In acute lesions tPA co-localized with Fibrin (Ogen) on large diameter Axons also stained with SMI-32, an ImmunoHistoChemical marker of Axonal Damage

The uPA-uPAR complex, concentrated on inflammatory cells in the PeriVascular zone of the evolving lesion, may facilitate cellular infiltration into the CNS which is amplified by MMP- mediated degradation of blood vessel Matrix.

tPA localization on injured Axons may be a marker of Axonal Damage or represent a protective mechanism aimed at removal of Fibrin deposits and restoration of Axonal function.

Multiple Sclerosis is characterized Morphologically by the key features: DeMyelination, Inflammation, Gliosis and Axonal damage.

In recent years, it has become more evident that Axonal damage is the major Morphological substrate of permanent clinical disability.

In our study, we investigated the occurrence of acute Axonal damage determined by ImmunoCytoChemistry for Amyloid Precursor Protein (APP) which is produced in Neurons and accumulates at sites of recent Axon transection or damage.

The numbers of APP-positive Axons in Multiple Sclerosis lesions were correlated with the disease duration and course.

Most APP-positive Axons were detected within the first year after disease onset, but acute Axonal damage was also detected to a minor degree in Lesions of patients with a disease duration of 10 years and more.

This effect was not due to the lack of active DeMyelinating lesions in the chronic disease stage. Late ReMyelinated lesions (so-called shadow plaques) did not show signs of Axon destruction.

The number of inflammatory cells showed a decrease over time similar to that of the number of APP-positive Axons.

There was a significant correlation between the extent of Axon Damage and the numbers of CD8⁺ CytoToxic T-Cells and Macrophages/Microglia.

Our results indicate that a putative Axon-protective treatment should start as early as possible and include strategies preventing T-Cell/Macrophage-mediated Axon destruction and leading to ReMyelination of Axons.

Recent Magnetic Resonance (MR) studies of Multiple Sclerosis lesions indicate that Axonal injury is a major correlate of permanent clinical deficit. In the present study we systematically quantified acute Axonal injury.

Defined by ImmunoReactivity for ß-Amyloid-Precursor-Protein in dystrophic Neurites, in the Central Nervous System of 22 Multiple Sclerosis patients and 18 rats with Myelin-Oligodendrocyte Glycoprotein (MOG)-induced chronic Autoimmune Encephalomyelitis (EAE).

The highest incidence of acute Axonal injury was found during active DeMyelination, which was associated with Axonal damage in PeriPlaque and in the Normal-Appearing White Matter of actively DeMyelinating cases.

In addition, low but significant Axonal injury was also observed in inactive DeMyelinated plaques. In contrast, no significant Axonal damage was found in ReMyelinated shadow plaques.

The patterns of Axonal pathology in chronic active EAE were qualitatively and quantitatively similar to those found in Multiple Sclerosis.

Our studies confirm previous observations of Axonal destruction in Multiple Sclerosis lesions during active DeMyelination, but also indicate that ongoing Axonal damage in inactive lesions may significantly contribute to the clinical progression of the disease.

The results further emphasize that MOG-induced EAE may serve as a suitable model for testing Axon-protective therapies in Inflammatory DeMyelinating conditions.