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, 7 (8), e43484

Pathological Changes in the White Matter After Spinal Contusion Injury in the Rat

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Pathological Changes in the White Matter After Spinal Contusion Injury in the Rat

C Joakim Ek et al. PLoS One.

Abstract

It has been shown previously that after spinal cord injury, the loss of grey matter is relatively faster than loss of white matter suggesting interventions to save white matter tracts offer better therapeutic possibilities. Loss of white matter in and around the injury site is believed to be the main underlying cause for the subsequent loss of neurological functions. In this study we used a series of techniques, including estimations of the number of axons with pathology, immunohistochemistry and mapping of distribution of pathological axons, to better understand the temporal and spatial pathological events in white matter following contusion injury to the rat spinal cord. There was an initial rapid loss of axons with no detectable further loss beyond 1 week after injury. Immunoreactivity for CNPase indicated that changes to oligodendrocytes are rapid, extending to several millimetres away from injury site and preceding much of the axonal loss, giving early prediction of the final volume of white matter that survived. It seems that in juvenile rats the myelination of axons in white matter tracts continues for some time, which has an important bearing on interpretation of our, and previous, studies. The amount of myelin debris and axon pathology progressively decreased with time but could still be observed at 10 weeks after injury, especially at more distant rostral and caudal levels from the injury site. This study provides new methods to assess injuries to spinal cord and indicates that early interventions are needed for the successful sparing of white matter tracts following injury.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Plastic sections (0.5 µm) stained with methylene blue of aged matched control and at 10 weeks after injury rat spinal cords.
(A) dorsal column, (B) ventrolateral tracts at different distances from injury centre. The #-symbol marks the cyst that develops after the injury. In the dorsal column at 10 weeks after injury only a small area is left in which axons are visible (area marked with dotted line); this area becomes progressively larger rostrally. Caudal to the injury the dorsal column is little affected; however, the corticospinal tract that runs at the base of the dorsal column (marked with an asterix) is not visible caudal to the injury but in contrast appears normal rostrally. Scale bars are 300 µm.
Figure 2
Figure 2. Area of CNPase immunoreactivity in tissue sections of spinal cords in control and injured animals.
A) Bar graphs represent average positive area (mm2) in tissue sections (12–14 per animal) between 5 mm rostral to 5 mm caudal of injury centre (±SEM, n = 3–4 animals per group). Open bars are age matched controls and closed bars are injury groups. In controls the CNPase area progressively increased with age of rats from around 1.4 mm2 (24 hours sham controls) to 2.2 mm2 (10 weeks after injury). At 2 hours the area was slightly lower than 24 hours sham controls but was significantly lower at all later stages in the injury group cords, however, no significantly difference was found between 24 hours and 10 weeks after the injury. 2 h – 2 hours, 24 h – 24 hours, 4 d – 4 days, 4 w - 4 weeks, 10 w – 10 weeks. B) CNPase area at different distances from centre of injury. At 2 hours after the injury the area is similar to controls except around middle of the injury (approximately1 mm on either side) where it is lower. In all other injury groups the CNPase area is well below controls in the centre of injury and up to 4–5 mm caudal and appears somewhat lower even up to 5 mm rostral to injury. Data are mean ± SEM.
Figure 3
Figure 3. Illustrations of CNPase immunoreactivity in 24-hour sham controls, 2-, 24- and 10-weeks after injury.
In control animals immunoreactivity is mostly confined to white matter of spinal cord. At 2 hours after injury immunoreactivity is lost in the dorsal column and the apex of ventromedial parts of white matter in the centre of injury. At 24 hours after injury, nearly all immunoreactivity is confined to an outer rim of the cord in the centre of injury. Immunoreactivity extends further towards the middle of the cord away from the injury site and at 5 mm rostral/caudal the pattern is similar to control cords. At 10 weeks after the injury only a small rim of tissue is left in the centre of the injury and nearly all this tissue is CNPase immunoreactive. At 5 mm caudal, the pattern of staining is similar to controls and likewise at 5 mm rostral expect for little immunoreactivity in the most medial parts of the dorsal column. Scale bar is 1 mm.
Figure 4
Figure 4. Illustrations of Luxol Fast Blue (LFB) staining of paraffin sections at different times after injury along with 24 hour and 10 week sham controls.
At 2 hours after injury LFB staining appears similar to sham controls animals in white matter although blood clots also stain positive for LFB; these can be seen up to 4 days after the injury making it more difficult to interpret the results from these times. At 24 hours the LFB positive area is greatly reduced and in the centre of injury there is faint staining on the outer edges of the ventral white matter (similar to CNPase staining, see Fig. 5). At 4 and 10-weeks after the injury, most of the tissue that is left is LFB positive Note also the larger LFB positive area in 10-week shams compared to 24-hour shams.
Figure 5
Figure 5. Number of myelinated axons in the dorsal column (A) and ventrolateral tracts (B) in control and spinal injured rats.
A) In the middle of the injury site at 24 hours after the injury, the number of axons is reduced to 23% of controls (0 week) and further reduced to 4–6% at later times after injury. The number of axons was similar in animals at 1–10 weeks after the injury at all distances from injury and was much lower than controls even at 9 mm rostral to injury (43–52% of 0 week controls) whereas at 9 mm caudal numbers were similar to controls. Note that in 10-week controls the number of axons is about 38% (or 12,000 axons) more than in 0-week controls in the dorsal column. B) In the middle of the injury site at 24 hours after the injury, the number of axons is reduced to 54% of controls (0 week) and further reduced to 13–17% at later times after injury. The number of axons was similar in animals at 1–10 weeks after the injury at all distances from injury and was lower than controls at 9 mm rostral (71–83% of 0 week controls) whereas at 9 mm caudal it was similar to controls. Note that in 10 week controls the number of myelinated axons is about 40% (or 76,000 axons) more than in 0 week controls in ventrolateral tracts.
Figure 6
Figure 6. Area of myelinated fibres (closed circles, left y-axis) and axon density (closed triangles, right y-axis) at different times after the injury (x-axis).
Open symbols represent control animals.
Figure 7
Figure 7
A) Number of axons with moderate pathology at 1-, 4- and 10-weeks after SCI in dorsal tracts (DT) and ventrolateral tracts (VLT). Numbers in brackets are axons exhibiting severe pathology. Note: These numbers should not be regarded as absolute numbers but rather as indicative of relative differences (see Methods). Examples of pathology are shown on right. Data are mean ± SEM (n = 3–4 at each time). B) Number of empty/collapsed myelin sheaths (examples shown on right) at 1-, 4- and 10-weeks after SCI in dorsal tracts (DT) and ventrolateral tracts (VLT). Data are mean ± SEM (n = 3–4 at each time).
Figure 8
Figure 8. Illustrations of the general distribution of mapped axonal pathology at 1 week (A–D) and 10 weeks (E–H) after injury.
Axons with moderate pathology are shown with red circles and axons with severe pathology with yellow stars. For illustrations of pathology see Figure 7. A) Axons in the dorsal column, 9 mm rostral of injury. Note that the pathology is uniformly spread throughout the white matter, outside the central core that is filled with degenerated axons (area inside dashed line). B) Axons in the dorsal column, 9 mm caudal of injury. Axon pathology is visible almost exclusively in the dorsolateral parts of the column where the axons enter from the spinal roots. C) Axons in the ventrolateral tracts, 9 mm rostral of injury. Axons pathology is uniformly spread within the white matter with some concentration to the medial parts. D) Axons in the ventrolateral tracts, 9 mm caudal of injury. Axon pathology is mostly restricted to the outer layers of the white matter close to the pial rim. Note that the numbers are much less than rostral to the injury. E) Axons in the dorsal column, 9 mm rostral of injury. A few axons are found outside the central core that is filled with degenerated axons (area inside dashed line). F) Axons in the dorsal column, 9 mm caudal of injury. A few axons are found throughout the column. G) Axons in the ventrolateral tracts, 9 mm rostral of injury. Axons pathology is rather uniform within the white matter. H) Axons in the ventrolateral tracts, 9 mm caudal of injury. Axon pathology is concentrated towards the pial rim in white matter and numbers are similar to caudal to injury (G).

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References

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Grant support

This work was supported by the Victorian Neurotrauma Initiative (Grant #DP048). For more information about this research fund, go to http://www.vni.com.au/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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