A large animal neuropathic pain model in sheep: a strategy for improving the predictability of preclinical models for therapeutic development

Abstract

Background: Evaluation of analgesics in large animals is a necessary step in the development of better pain medications or gene therapy prior to clinical trials. However, chronic neuropathic pain models in large animals are limited. To address this deficiency, we developed a neuropathic pain model in sheep, which shares many anatomical similarities in spine dimensions and cerebrospinal fluid volume as humans.

Methods: A neuropathic pain state was induced in sheep by tight ligation and axotomy of the common peroneal nerve. The analgesic effect of intrathecal (IT) morphine was investigated. Interspecies comparison was conducted by analyzing the ceiling doses of IT morphine for humans, sheep, and rats.

Results: Peroneal nerve injury (PNI) produced an 86% decrease in von-Frey filament-evoked withdrawal threshold on postsurgery day 3 and the decrease lasted for the 8-week test period. Compared to the pre-injury, sham, and contralateral hindlimb, the IT morphine dose that produces 50% of maximum analgesia (ED(50)) for injured PNI hindlimb was 1.8-fold larger and E(max), the dose that produces maximal analgesia, was 6.1-fold lower. The sheep model closely predicts human IT morphine ceiling dose by allometric scaling. This is in contrast to the approximately 10-fold lower morphine ceiling dose predicted by the rat spinal nerve ligated or spared nerve injury models.

Conclusion: PNI sheep model has a fast onset and shows stable and long-lasting pain behavioral characteristics. Since the antinociceptive properties of IT morphine are similar to those observed in humans, the PNI sheep model will be a useful tool for the development of analgesics. Its large size and consistent chronic pain behavior will facilitate the development and evaluation of surgical intervention and gene therapy. The PNI sheep pain model provides us with the opportunity for multi-species testing, which will improve the success of clinical trials.

Keywords: interspecies drug scaling; neuropathic pain model; ovine; ovine pain model.

Figure 1

Behavioral test area in sheep. (A) Sheep in a stanchion. The sheep was able to move forward and backward within the limited space of the stanchion. The testing area (circled in red) is located below the patella and above the tuber calcanei. (B) Cutaneous innervation of the test area. Notes: The cutaneous innervation of the sural nerve is located on the lateral side of the hindlimb. The test area (circled in red) is between the bony landmarks of the patella and tuber calcanei and within the areas of innervation of the lateral (shaded with dots) and caudal cutaneous (shaded with diagonal lines) sural nerves.

Figure 2

Sheep hindlimb withdrawal thresholds. Notes: In pre-injury sheep, withdrawal thresholds were determined 1–2 weeks prior to surgery (n = 6). There was no significant difference between ipsilateral and contralateral hindlimbs. In sham sheep, withdrawal thresholds of ipsilateral and contralateral sham hindlimbs, determined 3 weeks after sham surgery, were not significantly different (n = 3). Furthermore, there was no significant difference between sham and pre-injury sheep. In nerve injured sheep, withdrawal thresholds of ipsilateral and contralateral hindlimbs were measured 3 weeks after PNI surgery (n = 6). Data were analyzed using one-way ANOVA followed by Bonferroni correction. The ipsilateral nerve injured hindlimb showed a markedly reduced threshold (9.6 ± 1.7 g, n = 6); *P < 0.0001. The withdrawal threshold of the contralateral hindlimb was not significantly different from sham or pre-injury sheep. Abbreviations: ANOVA, analysis of variance; PNI, peroneal nerve injury.

Figure 3

Development of prolonged allodynia after nerve injury. Hindlimb withdrawal thresholds were determined over the 8-week test period (n = 6). (A) Withdrawal thresholds of the contralateral hindlimb showed no significant difference in sham and PNI sheep. (B) Withdrawal thresholds of the ipsilateral hindlimb of PNI sheep were significantly reduced 3 days after nerve injury and remained reduced for the 8 weeks of the study. Notes: Withdrawal thresholds of the ipsilateral hindlimb of sham sheep were not significantly different from those of the contralateral hindlimb and remained unchanged for the duration of the study. Comparison of withdrawal thresholds among sheep groups was analyzed by mixed ANOVA. *Significant difference (P < 0.001) from day 0. All of the experiments were repeated in six sheep. Abbreviations: ANOVA, analysis of variance; PNI, peroneal nerve injury.

Figure 4

Time course of IT morphine effect. Notes: The analgesia produced by a single dose of IT morphine was measured in pre-injury sheep (n = 3). A dose of morphine, eg, 50 μg/100 μL, was given at time 0. The withdrawal thresholds were measured at various intervals. The analgesia could be observed within 15 minutes of morphine application and reached a peak value after 30 minutes. The analgesia was maintained at the peak level for another 45 minutes before beginning to decrease at 75 minutes after morphine application. The morphine analgesic effect dissipated in 150–200 minutes. The level of peak analgesia was dose-dependent. Data were analyzed by one-way ANOVA repeated measures followed by post hoc test using the Bonferroni method. *Significant change from the withdrawal threshold measured prior to morphine injection. Abbreviations: ANOVA, analysis of variance; IT, intrathecal.

Figure 5

Dose-response of IT morphine on hindlimb withdrawal threshold. The hindlimb withdrawal threshold was determined 30 minutes after each IT dose of morphine. Each dose of morphine was given in 100 μL volume. Pre-injury sheep were tested 1–2 weeks before surgery. Sham and PNI sheep were tested 3 weeks after surgery. (A) Dose–response curve of pre-injury, sham, and nerve-injured contralateral hindlimbs. Dose–response curves were plotted as withdrawal threshold (g) versus cumulative morphine dose. The theoretical curves (solid lines) shown were fit to the contralateral nerve injured data with the Hill equation. The ED₅₀ was determined from the fitted curve. The morphine ED₅₀ for contralateral hindlimbs of pre-injury (73.4 μg), sham (74.8 μg), and nerve-injured sheep (88.3 μg) were not significantly different. (B) Dose–response curve of pre-injury, sham, and nerve-injured ipsilateral hindlimbs. The ipsilateral pre-injury curve was extrapolated to the x-axis limit. The theoretical curves (solid lines) shown were fit to the ipsilateral pre-injury and ipsilateral nerve-injured data with the Hill equation. Note: The ED₅₀ was 76.3 μg for pre-injury, 65.5 μg for sham, and 159.0 μg for nerve-injured sheep. Some of the standard errors of the data ≤ symbol sizes. Abbreviations: ED₅₀, the IT morphine dose that produces 50% of maximal analgesia; IT, intrathecal; PNI, peroneal nerve injury.