Pathological Studies

Axon Pathology

Although the MS lesion includes both inflammatory and demyelinating components, their relative influence on axonal loss is unclear. Classical neuropathologic descriptions of Charcot (1880), Marburg (1906), and Doinikow (1915) recognized degeneration of axons in MS lesions, but emphasized the primary demyelinating nature of the disease (105). More recent studies have demonstrated a high incidence of acute axonal injury within both chronic and early MS lesions (106-108), although the extent of axonal injury is variable, ranging from 20% to 90% reduction in axonal density relative to the periplaque white matter (Figure 10). Furthermore, there is interindividual heterogeneity in the extent of acute axonal injury (27). Pathological studies reveal that myelin and axonal pathology may occur independent of one another. Ongoing axonal injury is present in inactive plaques, and the damage to axons does not seem to depend on the stage of demyelinating activity (27). Furthermore, acute axonal injury can be found in the normal appearing and periplaque white matter of MS patients.

Data are limited on the mechanisms of axonal injury (Figure 11). The extent of axonal transection in early active lesions correlates with inflammation, therefore

Figure 10 (See color insert.) Axon loss in multiple sclerosis. Axonal density is reduced at the plaque edge and the plaque center, relative to the PPWM (A; neurofilament protein). Some of the reduced neurofilament staining at the active plaque edge can be attributed to macrophage infiltration. Consistent with acute axonal injury are numerous enlarged axonal profiles, axonal spheroids, and fragmented axons within the lesion (B, amyloid precursor protein; C, Bielschowsky silver). Abbreviation: PPWM, periplaque white matter.

Figure 10 (See color insert.) Axon loss in multiple sclerosis. Axonal density is reduced at the plaque edge and the plaque center, relative to the PPWM (A; neurofilament protein). Some of the reduced neurofilament staining at the active plaque edge can be attributed to macrophage infiltration. Consistent with acute axonal injury are numerous enlarged axonal profiles, axonal spheroids, and fragmented axons within the lesion (B, amyloid precursor protein; C, Bielschowsky silver). Abbreviation: PPWM, periplaque white matter.

Figure 11 (See color insert.) Mechanisms of axonal destruction: phases and effector mechanisms of axonal degeneration during active demyelination and in inactive demyelinated lesions. The final pathway of axon destruction is due to mitochondrial dysfunction, ion influx into the axon, and activation of proteases. Source: From Ref. 163.

Figure 11 (See color insert.) Mechanisms of axonal destruction: phases and effector mechanisms of axonal degeneration during active demyelination and in inactive demyelinated lesions. The final pathway of axon destruction is due to mitochondrial dysfunction, ion influx into the axon, and activation of proteases. Source: From Ref. 163.

during the acute stages of the disease, inflammatory mediators likely contribute to axonal injury. An association between the numbers of CD8+ T-cells and the extent of axon damage has been reported (27), and experimental studies implicate a CD8-MHC class I mediated pathway of axon destruction (103). Furthermore, the attachment of activated CD8+ T-cells containing cytotoxic granules polarized toward the demyelinated axon suggest direct CD8+ T-cell mediated cytotoxicity (26). Macrophages and microglia are often found in close contact with degenerating axons. Toxic inflammatory mediators liberated from these cells, such as proteases, cyto-kines, and free radicals, including nitric oxide (NO), may also lead to axonal injury. At low concentrations, NO induces a functional conduction block, but at higher concentrations, NO derivatives may irreversibly damage axons, particularly when they are electrically active (109). Axon-specific antibodies and complement may also be involved in mediating axonal injury. Anti-ganglioside antibodies were found to be significantly higher in PPMS than in SPMS or RRMS (110). Axons exposed to complement after demyelination may directly activate the complement cascade (111).

The magnitude of axonal loss in chronic lesions suggests mechanisms other than inflammatory demyelination may contribute to axonal damage during these later disease phases. Extensive acute axonal injury occurs during early stages of demyelination; however, a slow ongoing axonal destruction is also present in inactive MS lesions that lack inflammation (106). Although only a few axons are destroyed at a given time point, such lesions may persist in the CNS for years. In addition, repeated demyelination within previously remeylinated lesions may contribute to axonal loss in chronic MS (50). Chronically demyelinated axons may also degenerate due to the lack of trophic support from myelin and oligodendrocytes. Mice lacking certain myelin proteins (MAG and PLP) demonstrate late onset axonal pathology, as well as evidence for an increased incidence of Wallerian degeneration (112,113). Secondary (Wallerian) degeneration also contributes to diffuse axonal loss (114).

The mechanisms of axonal destruction in MS may vary depending on the phase of the disease. In early phases axonal injury correlates with inflammation, whereas during later phases this correlation is less evident. This might explain the benefit of anti-inflammatory and immunomodulatory agents on early relapsing MS, with limited, if any, benefit on gradual disease progression.

Once axonal injury has been triggered, a cascade of downstream mechanisms ultimately leading to axonal disintegration occurs (114). These mechanisms are similar in a variety of pathologic conditions including inflammation, ischemia, and trauma. Acute axonal injury leads to a disturbance in the axoplasmic membrane permeability and subsequent energy failure leading to uncontrolled sodium influx into the axoplasm, which reverses the sodium/calcium exchanger and results in excess intraxonal calcium. This activates Ca2+-dependent proteases, which degrade cytoskeletal proteins, further impairing axonal transport. Voltage gated calcium channels (VGCC) accumulate at sites of disturbed axonal transport, leading to further Ca2+ influx, and eventually dissolution of the axonal cytoskeleton and axo-nal disintegration. Therapeutic strategies that inhibit different steps in the execution phase of axonal destruction, such as Na+ channel blockers, inhibitors of the Na+-Ca2+ exchanger, blockade of VGCCs, or inhibition of calcium-dependent proteases, may help limit axonal destruction in MS. Clinical trials are needed with these agents to determine whether they slow disease progression.

Gray Matter Pathology

By concentrating on focal white matter lesions, previous neuropathological studies have not found major differences between patients with relapsing or progressive disease (115). However, there are pathological alterations in both the gray matter and NAWM of MS patients who contribute to disability.

MS may involve the gray matter, either as a classically demyelinated plaque or as neuronal loss and atrophy following retrograde degeneration from white matter lesions (3). Demyelinated plaques may be found in deep cerebral nuclei (116), or in the cerebral cortex (3). Cortical plaques are a well recognized but variable feature of MS pathology. Three types of cortical lesions can be distinguished: intracortical perivascular lesions; cortico-subcortical compound lesions affecting gray and white matter; and surface oriented band-like cortical lesions (117-120). The first two types develop around small veins and venules, whereas the third type is characterized by demyelination of the outer three to five layers of the cortex, resulting in band-like demyelinated lesions spanning several millimeters or centimeters of the cortical surface (118). This latter cortical lesion is the most common. These cortical lesions have a predilection for the cortical sulci, as well as the cingulate, temporal, insular, and cerebellar cortex. The lesions are associated with inflammatory infiltrates in the meninges.

Although cortical plaques share some pathologic features with white matter plaques, including demyelination, relative axonal and neuronal preservation, and some remyelination, they differ in several fundamental respects (118). The lesions tend to be less inflammatory, and blood-brain barrier damage is negligible, even when the lesions are in the stage of active demyelination. A quantitative study of Tand B-cell infiltrates showed no significant differences between the normal cortex of control patients and those with MS or demyelinated lesions in the cortex (119). However, cortical plaques are associated with massive activation of cortical microglia (119), and very high expression levels of i-NOS. Cortical lesions tend to be associated with less tissue destruction, likely due to the limited amount of myelin, coupled with the limited axonal and neuronal injury.

The degree of cortical involvement and whether it correlates with clinical course or disability in MS are unknown. Cortical demyelination could impact neuronal, dendritic, and axonal function, viability, and survival. A recent study demonstrated the presence of apoptotic neurons within the demyelinated cortex (118). This may be relevant to the pathogenesis of neurologic and cognitive disability in MS and could, in part, explain why the disease progresses in PPMS in the absence of extensive white matter abnormalities. Degeneration of cortical neurons could also partly explain the diffuse NAA loss observed in the NAWM of PPMS patients. Furthermore, cortical damage could lead to secondary tract degeneration, which may account for some of the diffuse spinal cord changes observed in PPMS. Besides demyelination, the cerebral cortex of MS patients may also be affected by tissue loss and atrophy, particularly at sites of severe focal or diffuse white matter injury. Neurons in such lesions may show signs of retrograde reaction, such as central chromatolysis. Quantitative MRI analyses show that cortical atrophy may occur early and to some extent predicts the clinical course and the development of cognitive impairment (121). Furthermore, degeneration of cortical neurons could contribute to the diffuse NAA loss described within both the NAWM and spinal cord. Recent observations suggest that patients with SPMS and PPMS contain a larger number of cortical lesions as compared to RRMS (122). These observations may explain why the disease progresses in PPMS in the absence of extensive white matter abnormalities.

NAWM Pathology

Previous studies have been limited in their ability to correlate functional neurological deficit with focal white matter lesions determined by quantitative MRI techniques. This is particularly the case in PPMS patients, in which severe neurological deficits are associated with a surprisingly low lesion load in the brain (101,123) and spinal cord (124,125). Although diffuse NAWM injury is in part due to axonal transection within plaques leading to secondary (Wallerian) degeneration, recent MRI data indicate that extent of tissue damage within focal plaques does not fully explain the degree of diffuse white matter changes (126,127), but suggest that global permanent neurological deficit may be determined by global and diffuse changes in NAWM (98,128-130).

There are few pathological studies of the NAWM in MS. Many have described mild inflammation (mainly CD8+ T-cells), microglial activation, gliosis, increased expression of proteolytic enzymes within astrocytes and microglia, diffuse axonal injury, and nerve fiber degeneration (106,131-135). A recent study compared the global brain damage in acute, relapsing, and progressive MS, and found a diffuse inflammatory process characterized by perivascular and paranechymal inflammatory infiltrates in progressive, but not acute or relapsing disease (122). The extent of inflammation was distributed globally throughout the brain, and was associated with widespread microglial activation characterized by CD68 expression, a marker for phagocytic activity, as well as MHC class II antigen and iNOS expression. Despite the lack of primary demyelination in the "normal" white matter, axonal spheroids and terminal axonal swellings were variably present throughout the tissue. The extent of inflammation and axonal injury in the NAWM, as well as the degree and character of cortical demyelination, did not correlate with the number, distribution, activity, or destructiveness of focal white matter lesions (122).

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