Assessment of functional and behavioral changes sensitive to painful disc degeneration

Abstract

The development of an in vivo rodent discogenic pain model can provide insight into mechanisms for painful disc degeneration. Painful disc degeneration in rodents can be inferred by examining responses to external stimuli, observing pain-related behaviors, and measuring functional performance. This study compared the sensitivity of multiple pain and functional assessment methods to disc disruption for identifying the parameters sensitive to painful disc degeneration in rats. Disc degeneration was induced in rats by annular injury with saline injection. The severity of disc degeneration, pain sensitivity, and functional performance were compared to sham and naïve control rats. Saline injection induced disc degeneration with decreased disc height and MRI signal intensity as well as more fibrous nucleus pulposus, disorganized annular lamellae and decreased proteoglycan. Rats also demonstrated increased painful behaviors including decreased hindpaw mechanical and thermal sensitivities, increased grooming, and altered gait patterns with hindpaw mechanical hyperalgesia and duration of grooming tests being most sensitive. This is the first study to compare sensitivities of different pain assessment methods in an in vivo rat model of disc degeneration. Hindpaw mechanical sensitivity and duration of grooming were the most sensitive parameters to surgically induced degenerative changes and overall results were suggestive of disc degeneration associated pain.

Keywords: disc degeneration; discogenic pain; functional performance; in vivo rat model; pain sensitivity.

Figure 1

(A) Schematic diagram showing surgical procedure with annular puncture at ventral portion of L3-4, L4-5, and L5-6 IVDs. (B) Representative T2-weighted MRI images of rat lumbar spines from näve, sham, and PBS groups. Injected IVDs showed decreased signal intensity (indicated with red circles). (C) Measurement of IVD height, which is defined as intervertebral space between vertebral boundaries (i.e., the yellow part with red boundary lines). (D) Representative radiographs of rat lumbar spines from näve, sham, and PBS groups. (E) The changes of injured IVD height (normalized to pre-surgery) of näve, sham, and PBS groups, the standard deviations are marked as error bars, and significant differences are indicated.

Figure 2

Safranin-O/Fast-Green/hematoxylin staining for rat lumbar discs from all experimental groups (A for naive, B for sham, and C for PBS). The AF regions (indicated with blue rectangles) are highlighted and shown at higher magnifications. The black arrow indicates the annular disruption during annular puncture.

Figure 3

(A) The experimental setup of the rotarod test. (B) The changes of the maximum end-speed (normalized to pre-surgery) of näve, sham, and PBS groups, the standard deviations are marked as error bars, and significant differences are indicated. All animal groups exhibited reduced performance with time and no difference between groups.

Figure 4

The experimental setup for (A) hindpaw mechanical hyperalgesia test and (B) hindpaw thermal hyperalgesia test. (C) The changes of paw withdrawal threshold (normalized to pre-surgery) of näve, sham, and PBS groups, the standard deviations are marked as error bars, and significant differences are indicated. (D) The paw withdrawal latencies of the näve and PBS groups (standard deviations are marked as error bars).

Figure 5

(A) The experimental setup for the assessment of spontaneous behavior. (B) The results of spontaneous behaviors (e.g., duration of grooming, immobilization and hindlimb standing, as well as number of “wet-dog shake”) of näve, sham, and PBS groups, the standard deviations are marked as error bars, and significant differences are indicated.