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, 5 (8), e12021

Spatio-temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord

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Spatio-temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord

C Joakim Ek et al. PLoS One.

Abstract

Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92+/-0.23 mm3, mean+/-SD, n=5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed.

Conflict of interest statement

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

Figures

Figure 1
Figure 1. Spinal impactor device and measurements of initial injury volume.
A&B) The linear motor of the impactor is mounted on a stereotaxic frame and a servo-controller unit used to deliver a precisely controlled impact to the exposed spinal cord of adult rats. The vertebral column is stabilised by clamps attached to the dorsal spines of the vertebrae either side of the laminectomy site to prevent movement of the vertebral column on impact. C) Schematic diagram of the impactor device in operation. The tip is lowered manually using the manipulator arm controls until it is just touching the surface of the dura mater exposed via a laminectomy (left). The tip withdraw height, acceleration rate, impact velocity, depth of tip penetration, deceleration rate and dwell time are preset by the user in the control software. The position of the tip during the course of each impact is recorded in real-time and graphically displayed to confirm the impact parameters (right). D) Vibrating microtome section (100 µm) through the centre of the lesion site in a spinal cord from an animal at 15 min after injury. The section has not been stained and was viewed under transmitted light. The areas of visibly damaged tissue and haemorrhage (brown colour) in each section were outlined and measured. Note the extensive damage and bleeding in the central grey matter of the cord and undamaged appearance of the surrounding white matter. The impactor tip strikes vertically down onto the dorsal columns (arrow) and penetrates to a preset depth of 2.0 mm below the surface of the dura mater. Scale bar is 1 mm. E) Lesion area measurements in serial sections (100 µm thick) spanning the entire injury site from animals subjected to a single impact injury at T10. The spinal cords were collected 15 min after injury and each line represents a single animal. The mean lesion volume was 1.92±0.23 mm3 (SD, n = 5) with a range of 1.52 to 2.10 mm3.
Figure 2
Figure 2. Morphology of injury at 24 hours and 1 week.
Methylene blue stained semi-thin sections (0.5 µm) of spinal cords at 24 hours (A) and 1 week (B) after injury, from 5 mm rostral (+) to 5 mm caudal (−) of the centre of the injury site. At 24 hours damage was apparent within 2 to 3 mm of the centre of the injury site in both grey and white matter. No macrophages were visible in the tissue. At 1 week tissue was being cleared by numerous macrophages (*) that had infiltrated the tissue and the early stages of cyst formation can been seen at the centre of the injury. Scale bars are 1 mm.
Figure 3
Figure 3. Morphology of injury at 4 and 10 weeks.
Methylene blue stained semi-thin sections (0.5 µm) of spinal cords at 4 weeks (A) and 10 weeks (B) after injury, from 5 mm rostral (+) to 5 mm caudal (−) of the centre of the injury site. At 4 weeks a large cystic cavity was visible within the cord extending several millimetres along the cord. A small number of macrophages were present in the centre of the injury (*). A similar cystic cavity was present at 10 weeks, however, no macrophages were seen in the cord. Scale bars are 1 mm.
Figure 4
Figure 4. Measurement of spinal cord weight and area.
A) Weights (mg) of 10 mm spinal cord segments centred on the injury site. There was a significant reduction in weight compared to sham controls at 4 and 10 weeks after injury (p<0.001, ***). In injured animals, there was a significant decrease in weight between 4 days and 4 weeks after injury (p<0.01, **), but no further weight loss between 4 and 10 weeks after injury. Data are mean ± SEM, n = 6 for each group. B) Areas of spinal cord measured in H&E stained serial sections from 5 mm rostral to 5 mm caudal of the centre of the injury site at 24 hours, 4 days, 4 weeks and 10 weeks after injury. There was little difference in spinal cord area between 24 hours and 4 days, but a large reduction in area between 4 days and 4 weeks. There were no further reductions in area between 4 weeks and 10 weeks after injury. Data are mean ± SEM, n = 6 for each group.
Figure 5
Figure 5. NeuN immunoreactivity in tissue sections.
NeuN positive nuclei in spinal cord at 2-, 24-hours and 4 weeks after injury. At 2 hours (2 h), a small number of neurons were visible at the centre of the injury site located mainly in the dorsal horns. By 24 hours (24 h) there had been further loss of neurons up to 2 mm either side of the centre of the injury site. NeuN profiles at 4 weeks (4w) were similar to that at 24 hours indicating that the loss of grey matter neurons occurs within the first 24 hours (see also Fig. 6).
Figure 6
Figure 6. Counts of neurons in spinal cords.
A) Number of NeuN immunoreactive nuclei per section (mean ± SEM) in 10 mm segments centred on the injury site at different times after surgery (n = 3–4 animals for each group). For each animal, the average of 12–14 sections was determined. In sham-operated animals (open bars), there was little difference in number of neurons between 24 hours, 4 weeks and 10 weeks after surgery. In the injured animals, the number of neurons was significantly lower at 2 hours compared to sham controls (p<0.05, *) and was further reduced at 24 h (p<0.05, *). At 24 hours, 4 days, 4 weeks and 10 weeks, the number of neurons was significantly lower than in age matched sham controls (p<0.01) and compared to 2 h after injury (p<0.05). There was no significant change in the number of neurons from 24 hours to 10 weeks after injury. 2 h = 2 hours, 1d = 24 hours, 4d = 4 days, 4w = 4 weeks, 10w = 10 weeks. B) Number of NeuN neurons per section (mean ± SEM) in injured animals normalised to control animals at different distances from injury site (0 mm). At 2 hours after injury, the number of neurons was higher at each point along the cord compared to all later times after injury. There was little difference in neuron numbers at any point along the cord from 24 hours to 10 weeks after injury. Note the similar pattern of neuron loss rostral and caudal to the injury site. C) Number of grey matter neurons (immunoreactive for PGP9.5, NSE or NeuN) per section normalised to sham operated control animals in 10 mm segments centred on the injury site at different times after surgery. The similar neuron numbers from 24 hours to 10 weeks after injury indicate that the loss of grey matter neurons occurs within the first 24 hours. Data are mean ± SEM, n = 3–4 for each group.
Figure 7
Figure 7. Myelinated axons in spinal cords.
Stereological estimation of numbers of myelinated axons at the centre, 2 mm rostral and 2 mm caudal of injury site in control and injured animals. At 24 hours after injury, the number of axons at the centre of the injury site was reduced to 50% of control animals (open bars) and at 1–10 weeks was further reduced to 12–15% of controls. At 2 mm rostral to the centre of the injury site, the number of the number of axons was 85% of control animals at 24 hours after injury and 37–43% of controls at 1–10 weeks. At 2 mm caudal to the centre of the injury site, the number of axons was about 80% of control animals at 24 hours after injury and 30–45% of controls at 1–10 weeks. C  =  sham control, 1d = 24 hours, 1w = 1 week, 4w = 4 weeks, 10w = 10 weeks. Data are mean ± SEM, n = 3 for each group. *** = p<0.001.
Figure 8
Figure 8. Western blot analysis.
Analysis for GFAP, CNPase, NSE and PGP9.5 in 10 mm spinal cord segments centred on the injury site. Constant amount of protein (range 0.07–1.0 µg) for each marker was loaded into each well. All data are expressed as percentage change (± SEM) compared to sham operated age-matched control tissue. GFAP (specific for astrocytes) showed very large increases at 24 hours and 4 days after injury, which is a reflection of the activation of astrocytes in response to injury. GFAP levels were not significantly different from controls at 4 and 10 weeks after injury. In contrast, the levels of the NSE and PGP9.5 (specific for neurons) and CNPase (specific for oligodendrocytes) were reduced to 70% of controls at 24 hours after injury and to 30–40% up to 4 weeks. There were no further reductions up to 10 weeks after injury. Data are mean ± SEM, n = 6 for each group. Probability of significant difference compared to age matched sham operated controls: * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 9
Figure 9. Behavioural testing.
A) BBB scores for sham operated (open circles, n = 8) and spinal cord injured animals at 4 weeks (open triangles, n = 4) and 10 weeks (filled circles, n = 8–13) after injury. There was an improvement in BBB scores from 1 week up to 4 weeks after injury, but no further improvement up to 10 weeks. At all times after injury, the scores for the injured animals were significantly lower than for age-matched sham controls (p<0.001). Data are mean ± SEM. B) Number of errors (uses of the ledge by hindlimbs) in the ledged beam test by sham control (open circles, n = 8) and spinal cord injured rats at 4 weeks (open triangles, n = 4) or 10 weeks (filled circles, n = 8–13). The number of errors by the injured animals was significantly greater than sham controls at all time points (p<0.001). Data are mean± SEM. C) Number of errors (hindlimb foot slips) in the random rung ladder beam test in sham control (open circles, n = 4) and spinal cord injured rats at 10 weeks (filled circles, n = 4). The spinal cord injured animals made significantly more errors than age matched sham controls at each time point (p<0.001). Data are mean ± SEM.
Figure 10
Figure 10. Vascular supply after spinal cord injury.
A) Sagittal section (50 µm) of rat thoracic spinal cord showing the gross vascular anatomy. Note that parallel sulcal arteries, at every 50 to 100 µm along the cord, extending from the anterior spinal artery (arrowhead) into the central grey matter (*). This suggests a segmented vascular supply of the central grey matter. B–D) Sagittal sections (50 µm) of rat thoracic spinal cord 15 min after i.v. injection of a 70 kDa green fluorescent dextran into a rat 4 days after spinal injury. B) In this section from just outside the injury site, vessels are filled with the dextran indicating that blood flow is maintained in these vessels. C) Higher magnification shows that the dextran has reached small capillaries just outside the injury zone. This is in contrast to the injury site where capillaries are devoid of the dextran indicating that flow through these vessels is impeded (arrows in D). The highly segmental arrangement of the blood supply to the central grey matter (A) could account for the limited rostral and caudal expansion of the lesion since the blood supply to regions outside the initial injury zone appears to be maintained (B & C). Scale bars are 1 mm in A, 250 µm in B and 50 µm in C & D.

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