Axonal Loss in EAE Correlates with Inflammation and Disability

The use of animal models has provided valuable insights regarding the pathophysiological mechanisms and functional consequences of axonal injury during immunemediated demyelination. Axonal transection has been described in mouse (Brown et al., 1982; Craner et al., 2004), guinea pig (Raine and Cross, 1989), and primate (Raine et al., 1999; Mancardi et al., 2001) models of EAE as indicated by the presence of both axonal dystrophy and swellings.

Induction of EAE in SWXJ mice by immunization with the 139-151 peptide of PLP produces consistent progression from relapsing disease to chronic disability (Yu et al., 1996). The neurological disability or clinical score for each animal was correlated with inflammation and axonal loss at first attack and at end-stage disease (Wujek et al., 2002). Initiation of axonal pathology and progressive axonal loss in spinal cord white matter occurred early in the disease course and was detected on immunohistochemical examination (Wujek et al., 2002). Clinical scores correlated with spinal cord inflammation, but not axon loss, which is low after initial attack. In contrast, during the chronic end-stage of relapsing EAE, clinical scores correlated highly with axon loss in both cervical (P < 0.0001) and lumbar spinal cords (P < 0.004) (Fig. 5), but not inflammation. The end-stage mice with permanent limb paralysis had average spinal cord axonal losses of 59% at cervical and 43% at lumbar levels. The number of symptomatic relapses varied between one and five. Regression analysis verified a significant correlation between number of relapses and axonal loss, which was stronger for the cervical cord (p = 0.75) than for the lumbar cord (p = 0.63). The number of relapses for each mouse was significantly related to clinical score, as predicted from the relationship between episode number and axonal loss (Wujek et al., 2002).

Figure 5 Spinal cord axonal loss correlates with permanent neurological disability in mice with chronic EAE. Neurofilament-positive axons in dorsolateral region of spinal cord from control (A) and EAE mice with clinical scores of 0 (B), 2 (C), and 4 (D). Increasing axonal loss in cervical (white bars) and lumbar (black bars) spinal cord correlated with increasing clinical score (E). Asterisks indicate significant correlation between axon loss and clinical score. Spearman's rank correlation test was used to test for significance (cervical cord: p = 0.75; P = 0.0001; lumbar cord: p = 0.63; P = 0.004). (Reproduced from Wujek et al., 2002, with permission.)

Figure 5 Spinal cord axonal loss correlates with permanent neurological disability in mice with chronic EAE. Neurofilament-positive axons in dorsolateral region of spinal cord from control (A) and EAE mice with clinical scores of 0 (B), 2 (C), and 4 (D). Increasing axonal loss in cervical (white bars) and lumbar (black bars) spinal cord correlated with increasing clinical score (E). Asterisks indicate significant correlation between axon loss and clinical score. Spearman's rank correlation test was used to test for significance (cervical cord: p = 0.75; P = 0.0001; lumbar cord: p = 0.63; P = 0.004). (Reproduced from Wujek et al., 2002, with permission.)

A causal relationship between number of inflammatory attacks, axonal loss, and permanent neurological disability in mice with relapsing EAE is indicated and related to the stage of EAE by these data (Wujek et al., 2002). The correlation between clinical disability and the extent of axonal loss was poor after the initial exacerbation. During the stable chronic end-stage of the disease, however, mice showed signs of irreversible functional impairment of varying severity, which had a strong correlation with spinal cord axonal loss. For example, during the chronic EAE stage, relatively modest white-matter axon loss was detected in mice with minimal or no clinical signs, whereas extensive axonal loss was detected in the severely disabled animals. Specifically, in end-stage mice with no observable symptoms (clinical score 0), axonal loss was 30% in the cervical and 15% in the lumbar spinal cord compared to controls. Curiously, the amount of axonal loss was substantially higher than in first attack mice with a reversible clinical score of 4 (limb paralysis). The 43% to 59% axonal loss measured in irreversibly paralyzed chronic end-stage EAE mice is of similar extent to the 68% reduction in axons detected in paralyzed MS patient spinal cord lesions (Bjartmar et al., 2000). Mice with a clinical score of 2 exhibited poor righting reflex with the extent of axonal loss midway between the amounts of axonal loss detected in mice with scores of 0 and 4 (Fig. 5). Analysis of spinal cords collected from the most disabled end-stage mice identified some lesions consisting predominantly of damaged axons with extremely few normal axons (Wujek et al., 2002).

These data have two important ramifications for our understanding of neurological disability. First, these data indicate that acute reversible disability in relapsing EAE results from inflammation and edema not axonal transection, as functional disability did not strongly correlate with early axonal loss. Second, these data indicate that at chronic stages, when axonal degeneration highly correlates with disability, axonal loss is the cause of neurological disability in these EAE mice. Data from this animal model support the hypothesis that irreversible neurological disability occurs in MS once axonal loss surpasses a certain threshold. Suitable animal models will be required for the development of new neuroprotective drugs for MS. This relapsing EAE model recapitulates many pathological and functional aspects of MS. These mice also benefit from interferon-P treatment, exhibiting milder symptoms and fewer relapses (Yu et al., 1996). Finally, the spinal cord axonal loss measured in the chronic paralyzed mice is of similar magnitude as the axonal loss observed in spinal cord lesions of severely disabled patients with chronic MS (Ganter et al., 1999; Lovas et al.,

2000; Bjartmar et al., 2000). Thus, this particular EAE model may be useful for testing the efficacy of novel neuroprotective therapeutics being developed for patients with MS.

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