Inflammatory Processes Associated with Canine Intervertebral Disc Herniation

Abstract

Intervertebral disc herniation (IVDH) is an important pathology in humans and also in dogs. While the molecular disease mechanisms are well investigated in humans, little is known about the inflammatory mediators in naturally occurring canine IVDH. The objective of this study was to investigate whether the involved proinflammatory cytokines in human IVDH are also key cytokines in canine IVDH and thus to elucidate the suitability of the dog as a model for human trials. 59 samples from 25 dogs with surgically confirmed thoracolumbar IVDH were collected and classified in three subgroups: herniated (H), affected non-herniated (NH) disc, and adjacent non-affected (NA) disc. Discs from 11 healthy dogs acted as controls (C). Samples were analyzed for IL-1, IL-6, IL-8, and TNF-α expression (qPCR/ELISA) as well as cell infiltration and activation of the MAP kinase pathways (immunohistochemistry). Gene and protein expression of all key cytokines could be detected in IVDH affected dogs. Canine IVDH was significantly associated with a higher gene expression of IL-6 (H > C, NH > C) and TNF-α (H > C, NH > C, NA > C) and a significant down-regulation of IL-1β (H < C). Dogs with spontaneous pain had significantly higher IL-6 mRNA compared to those with pain arising only upon palpation. An inter-donor comparison (H and HN relative to NA) revealed a significant increase of IL-6 gene expression (H > NA, NH > NA). IL-8 (H > C, NA > C) and TNF-α (NH > C) protein levels were significantly increased in diseased dogs while inversely, IL-6 protein levels were significantly higher in patients with better clinical outcome. Aside from resident IVD cells, mostly monocytes and macrophages were found in extruded material, with concomitant activation of extracellular signal-regulated kinase p38 in the majority of samples. Dogs with spontaneous IVDH might provide a useful model for human disc diseases. Although the expression of key cytokines found in human IVDH was also demonstrated in canine tissue, the inflammatory mechanisms accompanying canine IVDH diverges partially from humans, which will require further investigations in the future. In dogs, IL-6 seems to play an important pathological role and may represent a new potential therapeutic target for canine patients.

Keywords: IL-1β; IL-6; IL-8; MAP kinase pathway; TNF-α; canine animal model; inflammatory mediators; intervertebral disc herniation.

Figure 1

Gene expression of IL-6 in herniated disc material, compared between dogs with differing grade of pain (n = 11) and shown as 2^−ΔCT. Grade 1 = no spontaneous pain, but discomfort upon palpation of the spine. Grade 2 = spontaneous pain and excessive pain upon palpation of the spine. Asterisks indicate statistical significance between groups at p < 0.05 (*).

Figure 2

Gene expression data (subgroup comparison, independent samples, n ≥ 6 per subgroup) of (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TNF-α, compared between herniated (H), non-herniated (NH), non-affected (NA) as well as healthy control (C) intervertebral disc material. Data are shown as 2^−ΔCT. Asterisks indicate statistical significance between groups at p < 0.05 (*) and p < 0.01 (**).

Figure 3

Fold change in IL-6 gene expression (interdonor comparison, n = 6 per subgroup) between each dog’s non-affected IVD material (NA) and the same dog’s herniated (H) or non-herniated (NH) intervertebral disc material. Data are shown as 2^−ΔΔCT. Asterisks indicate statistical significance between groups at p < 0.05 (*).

Figure 4

Protein expression of IL-6 in herniated disc material, compared betwen dogs with differing clinical outcome (n = 20) and shown as picogram of IL-6 per milligram total protein. Grade 0 = lack of improvement. Grade 1 = improvement of neurologic status, but not able to walk. Grade 2 = recovery of ambulation or at least one grade in neurologic grades 1 and 2. Asterisks indicate statistical significance between groups at p < 0.05 (*).

Figure 5

Protein expression data (subgroup comparison, independent samples, n ≥ 6 per subgroup) of (A) IL-1β, (B) IL-6, (C) IL-8, and (D) TNF-α, compared between herniated (H), non-herniated (NH), non-affected (NA) as well as healthy control (C) intervertebral disc material. Data are shown as picogram of each cytokine per milligram total protein. Asterisks indicate statistical significance between groups at p < 0.05 (*).

Figure 6

Immunohistochemical staining for extracellular signal-regulated kinase (ERK)1/2 of extruded canine intervertebral disc material (left). Expression of ERK (brownish coloration) was detected in the majority of the examined slides. On the right, negative control and positive control (canine mammar carcinoma) are depicted.