Spatio-temporal development of axonopathy in canine intervertebral disc disease as a translational large animal model for nonexperimental spinal cord injury

Abstract

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of β-amyloid precursor protein, non-phosphorylated neurofilament (n-NF) and growth-associated protein-43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n-NF. Real-time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)-2 and MMP-9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.

Figure 1

Semiquantitative analysis and H&E staining of swollen axons (A,C) and phagocytic microglia/macrophages (B,D) in the lesion epicenter. (A) Scatter plots with median illustrate the mean values of semiquantitative evaluation of swollen axons in H&E‐stained sections that are significantly increased in dogs with SCI compared with controls. (B) Increased numbers of phagocytic cells are exclusively noted in dogs with subacute SCI compared with controls and dogs with acute SCI (group 2). Group 1 = control animals. Group 2 = dogs with acute SCI. Group 3 = dogs with subacute SCI. Statistically significant differences are marked with an asterisk (Kruskal–Wallis test with subsequent pairwise Mann–Whitney U‐test, alpha adjusted according to the method of Bonferroni). (C) Swollen axons are a prominent finding in animals with SCI and are mainly located in the ventral funiculi of the white matter (epicenter of animal no. 20). (D) Phagocytic cells, mainly located in virtually empty and dilated myelin sheaths, are nearly completely restricted to animals with subacute SCI (group 3; epicenter of animal no. 23). Scale bar (C) = 50 μm (D) = 25 μm. H&E = hematoxylin and eosin; SCI = spinal cord injury.

Figure 2

Extension of axonal changes (A,B), phagocytic microglia/macrophages (C,D) and necrotic/haemorrhagic changes (E,F) in dogs with acute (group 2) and subacute (group 3) SCI. Minus signs on the X‐axis symbolize caudal (‐) and numbers without signs cranial direction, respectively. (A) Semiquantitatively evaluated swollen axons in dogs with acute SCI (group 2) are mainly located in the epicenter (0). One animal shows a significant spread of swollen axons over the entire investigated length of the spinal cord. (B) Axonal damage in dogs with subacute SCI (group 3) spreads over long distances with an accentuation to the caudal direction. (C) Phagocytic microglia/macrophages are restricted to a single animal with acute SCI. (D) In contrast, most dogs with subacute SCI show significant numbers of phagocytic microglia/macrophages extending over long distances from the epicenter (0). (E) Mild to moderate, multifocal hemorrhages and/or necrosis are seen in animals with acute SCI, mainly restricted to the epicenter and localization 1.5. (F) Some animals with subacute SCI showed mild, multifocal hemorrhages and/or necrosis, mainly restricted to the epicenter. SCI = spinal cord injury.

Figure 3

Immunohistochemistry of axonal changes in controls and the lesion epicenter of canine spinal cord injury (SCI). (A–C) Axonal expression of p‐NF. p‐NF is expressed by healthy axons in control animals (A) and additionally by swollen axons in dogs with acute (B) and subacute (C) SCI. (D–F) Axonal expression of n‐NF. n‐NF is not expressed in axons of control animals (D) whereas there is a significant n‐NF expression in swollen and normal‐diameter axons of dogs with acute (E) and subacute (F) trauma. (G–I) Axonal expression of β‐APP. β‐APP is confined to some glial cells and not expressed by healthy axons in control animals (G). In dogs with acute (H) and subacute (I) SCI β‐APP is mainly detected in swollen axons. (J–L) Axonal expression of GAP‐43. GAP‐43 is not expressed by healthy axons in control animals (J) whereas in dogs with acute (K) and subacute (L) trauma swollen axons expressing GAP‐43 are found within dilated myelin sheaths. Scale bar (A–L) = 100 μm. Avidin–biotin–complex method and staining dye 3,3′‐diaminobenzidine. β‐APP = β‐amyloid precursor protein; GAP = growth‐associated protein; n‐NF = non‐phosphorylated neurofilament; p‐NF = phosphorylated neurofilament.

Figure 4

Scatter plots with median illustrating the results of the quantitative immunohistochemical analysis of axonal changes in the lesion epicenter of canine SCI. Group 1 = controls, group 2 = dogs with acute SCI, group 3 = dogs with subacute SCI. Statistically significant differences are marked with an asterisk (Kruskal–Wallis test with subsequent pairwise Mann–Whitney U ‐test, alpha adjusted according to the method of Bonferroni). (A) The percentage of axons expressing n‐NF is increased in dogs with acute and subacute SCI compared with controls. (B) Depiction of the p‐NF/n‐NF ratio as determined by morphometrical assessment of the immunopositive total white matter. Because of the relative increase in n‐NF expression there is a decrease of the p‐NF/n‐NF ratio in dogs with acute and subacute SCI compared with controls. (C) The percentage of axons expressing β‐amyloid precursor protein is increased in dogs with SCI compared with controls. (D) Similarly, the percentage of axons expressing growth‐associated protein‐43 is increased in traumatized dogs compared with healthy controls. n‐NF = non‐phosphorylated neurofilament; p‐NF = phosphorylated neurofilament; SCI = spinal cord injury.

Figure 5

Transmission electron microscopy. (A) Intact axon of a control animal exhibiting mild myelin changes interpreted as fixation artifacts. (B) Myelin sheath dilatation and myelin fragmentation in an animal with acute SCI. (C) Axon swelling and axoplasmic degeneration with accumulation of numerous electron‐dense bodies (arrowheads; inset) and swollen mitochondria (arrows; inset) in an animal with subacute SCI. (D) Marked axoplasmic accumulation of mitochondria (arrows; inset) intermingled with numerous neurofilaments (inset) without dense body formation in a swollen axon of an animal with subacute SCI. (E) Immunoelectron microscopic illustration of potentially microtubule‐associated gold particles specifically labeling growth‐associated protein‐43 (arrow, inset) in a mitochondria‐rich swollen axon of the same animal as in (D). (F) Large phagocytic cell located within an empty myelin sheath showing phagocytosed myelin and axonal remnants in an animal with subacute SCI. A scale bar = 0.5 μm; B scale bar = 2.7 μm; C,D scale bar = 1.7 μm; E scale bar = 0.2 μm; F scale bar = 5.4 μm. SCI = spinal cord injury.

Figure 6

Scatter plots with median illustrating the results of the real‐time quantitative polymerase chain reaction for the analysis of normalised mRNA levels per 100 ng transcribed RNA of the gelatinases MMP‐2 and MMP‐9 in naturally traumatized dogs (A,B) and organotypic spinal cord slice cultures (C,D). Group 1 = controls, group 2 = dogs with acute SCI, group 3 = dogs with subacute SCI. 0, 3, 9 = days in culture. Statistically significant differences are marked with an asterisk [Kruskal–Wallis test with subsequent pairwise Mann–Whitney U ‐test, alpha adjusted according to the method of Bonferroni (A,B); Friedman test and subsequent pairwise signed rank test (C,D)]. (A) MMP‐2 mRNA is transiently downregulated in dogs with acute SCI compared with controls. (B) MMP‐9 shows a significant upregulation in dogs with acute SCI compared with controls. (C) In organotypic spinal cord slices MMP‐2 shows a delayed upregulation on day 9 compared with day 3 in culture. (D) MMP‐9 is upregulated on day 3 and 9 in cultivated spinal cord slices compared with the control tissue of 0 days. MMP = matrix metalloproteinases; SCI = spinal cord injury.