The rat intervertebral disk degeneration pain model: relationships between biological and structural alterations and pain

Abstract

Introduction: Degeneration of the interverterbral disk is as a cause of low-back pain is increasing. To gain insight into relationships between biological processes, structural alterations and behavioral pain, we created an animal model in rats.

Methods: Disk degeneration was induced by removal of the nucleus pulposus (NP) from the lumbar disks (L4/L5 and L5/L6) of Sprague Dawley rats using a 0.5-mm-diameter microsurgical drill. The degree of primary hyperalgesia was assessed by using an algometer to measure pain upon external pressure on injured lumbar disks. Biochemical and histological assessments and radiographs of injured disks were used for evaluation. We investigated therapeutic modulation of chronic pain by administering pharmaceutical drugs in this animal model.

Results: After removal of the NP, pressure hyperalgesia developed over the lower back. Nine weeks after surgery we observed damaged or degenerated disks with proteoglycan loss and narrowing of disk height. These biological and structural changes in disks were closely related to the sustained pain hyperalgesia. A high dose of morphine (6.7 mg/kg) resulted in effective pain relief. However, high doses of pregabalin (20 mg/kg), a drug that has been used for treatment of chronic neuropathic pain, as well as the anti-inflammatory drugs celecoxib (50 mg/kg; a selective inhibitor of cyclooxygenase 2 (COX-2)) and ketorolac (20 mg/kg; an inhibitor of COX-1 and COX-2), did not have significant antihyperalgesic effects in our disk injury animal model.

Conclusions: Although similarities in gene expression profiles suggest potential overlap in chronic pain pathways linked to disk injury or neuropathy, drug-testing results suggest that pain pathways linked to these two chronic pain conditions are mechanistically distinct. Our findings provide a foundation for future research on new therapeutic interventions that can lead to improvements in the treatment of patients with back pain due to disk degeneration.

Figure 1

Axial sections of the L4/L5 lumbar disks showing immunohistochemical staining with Safranin O, toluidine blue (nine weeks after surgery) and CD11b. (A) Safranin O-stained sections of sham control (left) and disk injury (right). (B) Toluidine blue-stained sections of sham control (top left) and traumatic disk puncture (top right) (original magnification, ×40). Lower panels show enlargements (original magnification, ×100) of boxed portions of top left and top right panels. Mast cells are indicated by dark blue staining. (C) CD11b-stained sections (original magnification, ×40). Immune cells are shown in dark blue staining. AF = annulus fibrosus; NP = nucleus pulposus.

Figure 2

Using digitized X-ray images, we analyzed measurements, including the vertebral body height and intervertebral disk height, with imaging software. (A) Digitized X-ray images of intervertebral disk segments obtained to compare presurgery (naïve), sham control and drill puncture. (B) Intervertebral disk height was calculated by averaging the measurements obtained from the anterior, middle and posterior portions of the intervertebral disk and dividing that average by the average adjacent vertebral body height. (C) The disk height index was measured nine weeks after surgery. The disk height index of disk degeneration (DDD) was significantly decreased compared to sham control (P < 0.01).

Figure 3

Behavioral pain assessments over a seven-week time period. Pressure hypersensitivity of the lower back was determined by measurement of the vocalization threshold to an applied force gauge (algometer). (A) Graph showing data derived from animals that underwent traumatic puncture by a drill (0.8 mm in diameter and 0.64 mm² area) (n = 12) compared with the sham surgery group (n = 12). *P < 0.001 versus sham group; †P < 0.001 versus presurgery baseline (time 0). (B) Graph showing data derived from animals punctured with a smaller drill (0.5 mm in diameter and 0.25 mm² area) (n = 6) were compared with the sham surgery group (n = 6). *P < 0.05 versus sham group; †P < 0.001 versus presurgery baseline; ‡P < 0.005 versus presurgery baseline.

Figure 4

Glial activity-associated gene analyses in rats with traumatic disk puncture-induced disc degeneration (DDD) by qRT-PCR and immunoblotting. (A) (a) through (c) Dorsal root ganglion. (B) (a) through (c) Spinal dorsal horn. (C) (a) and (b) α₂δ₁ expression in spinal dorsal horn. Values are means ± SEM.

Figure 5

Measurement of the vocalization threshold using a pressure algometer test to examine the pharmacological efficacy of drugs on traumatic disk puncture-initiated discogenic pain (tested during weeks 7 through 9). Administration of pregabalin (20 mg/kg), morphine (6.7 mg/kg), celecoxib (selective inhibitor of COX-2, 50 mg/kg), ketorolac (COX-1 and COX-2 inhibitor, 20 mg/kg) or control vehicle by either oral gavage or intraperitoneal injection followed by primary pressure pain assessment by algometer. Morphine demonstrated a potent analgesic drug effect with a single administration, whereas pregabalin, celecoxib and ketorolac use led to no significant behavioral changes compared to saline control. *P < 0.001 compared to the other drug groups; †P < 0.001 compared to presurgery baseline (time 0). COX-1 = cyclooxygenase 1; COX-2 = cyclooxygenase 2.