Evaluation of corticospinal axon loss by fluorescent dye tracing in mice with experimental autoimmune encephalomyelitis

Abstract

In both multiple sclerosis (MS) patients and experimental autoimmune encephalomyelitis (EAE) animals, axon loss has been demonstrated to correlate with neurological disability. However, it is difficult to accurately determine the location and severity of axonal damage since the lesion in MS or EAE is disseminated and is frequently in a relapsing-remitting mode. The corticospinal system is the only direct pathway from the motorsensory cortex to the spinal cord, and the major neural pathway for control of voluntary movement. Moreover, it is frequently involved in the pathological process of the disease. To evaluate corticospinal tract (CST) axon loss in EAE mice, we developed a direct tracing method with a fluorescent neuronal tracer DiI which was injected into the primary motor cortex and sensorimotor cortex to label the pyramidal neurons. The lesion location in the spinal cord and axon disruption were indicated by dye leakage. Using the EAE induced axon reduction as an index of the extent of axonal damage, our data showed a high correlation between the axonal loss and the behavioral outcome score in the EAE mice. The results were consistent with the axonal Bielschowsky silver staining. Thus, this CST tracing method permits monitoring of the axonal damage in EAE.

Figure 1. Course of functional deficient in the EAE mice

Data represent mean behavior scores measured daily for EAE animals. At 50 days after the first clinical onset, the animals were in the second attack courses.

Figure 2. Anterograde DiI-labeling of the CST

A schematic diagram shows the bilateral cortical injections for CST labeling (A). A small volume of DiI solution was delivered into the layer V of motor cortex through a finely drawn glass capillary with a microinjection system to label the pyramidal neurons (B) in a representative normal mouse. Fluorescent images show that the CST fibers were labeled by DiI one week after injection in a coronal section at the decussation level (C) and in longitudinal sections of cervical (D) and lumbar (E) enlargements. Scale Bars in B and C = 250 μm, in D and E = 25 μm.

Figure 3. Longitudinal Images show the CST damage in the spinal dorsal funiculus

A typical CST lesion site in the spinal cord was indicated with DiI leakage in a MCID image (A). The DiI-labeled axons were decreased caudal to the lesion site. In a single layer confocal image of the CST lesion site, blobs of leaked DiI show the axonal damage site (Arrows in B). In a more caudal lesion site, only few intact axons were labeled (C). The images are oriented with rostral at the top. Scale Bars in A = 250 μm, B and C = 25 μm.

Figure 4. Axonal quantification

DiI-labeled axon count was performed on the single layer confocal images of high cervical and lumbar cord (A). At the cervical level, the DiI-labeled axon number of EAE mice (B and D) was only slightly decreased compared with normal mice (B and C), while the EAE lesion significantly reduced the DiI-labeled intact axon at the lumbar level (B, E and F). Scale Bars in C-F = 50 μm.

Figure 5. Bielschowsky silver staining

CST axons in the ventral-most part of the spinal dorsal funiculus were stained on coronal paraffin sections of the cervical and lumbar cord in normal (A and C) and EAE mice (B and D). There was no significant decrease of cervical CST number in the EAE mice, while an average loss of 49.4% of CST number in the lumbar cord after EAE was estimated with Bielschowsky silver staining. Scale Bar = 50 μm.

Figure 6. Correlation between the axonal loss and functional deficient

Significant correlations between the axonal damage and behavior score in individual EAE mice at 50 days after the first attack were demonstrated with both DiI tracing method (quadrangle, r=0.80, p<0.05) and Bielschowsky silver staining (circle, r=0.77, p<0.05).