Development of a Phantom Limb Pain Model in Rats: Behavioral and Histochemical Evaluation

Abstract

Therapeutic strategies targeting phantom limb pain (PLP) provide inadequate pain relief; therefore, a robust and clinically relevant animal model is necessary. Animal models of PLP are based on a deafferentation injury followed by autotomy behavior. Clinical studies have shown that the presence of pre-amputation pain increases the risk of developing PLP. In the current study, we used Sprague-Dawley male rats with formalin injections or constriction nerve injury at different sites or time points prior to axotomy to mimic clinical scenarios of pre-amputation inflammatory and neuropathic pain. Animals were scored daily for PLP autotomy behaviors, and several pain-related biomarkers were evaluated to discover possible underlying pathological changes. Majority displayed some degree of autotomy behavior following axotomy. Injury prior to axotomy led to more severe PLP behavior compared to animals without preceding injury. Autotomy behaviors were more directed toward the pretreatment insult origin, suggestive of pain memory. Increased levels of IL-1β in cerebrospinal fluid and enhanced microglial responses and the expression of NaV1.7 were observed in animals displaying more severe PLP outcomes. Decreased expression of GAD65/67 was consistent with greater PLP behavior. This study provides a preclinical basis for future understanding and treatment development in the management of PLP.

Keywords: GAD65/67; Nav 1.7; autotomy; axotomy; phantom limb pain.

Figure 1

Models to induce PLP-like behavior in rats. (A) Injection of saline/formalin into the lateral plantar hindpaw 2 h prior to axotomy (lateral injection). (B) Injection of saline/formalin into the medial plantar hindpaw 2 h prior to axotomy (medial injection). (C) CCI at 1 day or 4 weeks prior to axotomy (CCI).

Figure 2

Autotomy onset and progression (A) and autotomy scores of individual digits (B) after lateral injection prior to axotomy. *P < 0.05, **P < 0.01 between saline- and formalin-pretreated groups. Vertical marks indicate the mean onset and cutoff times in each group.

Figure 3

Time course of PLP development as indicated by autotomy scores (A) and fraction of animals remaining (B) over time after lateral injection prior to axotomy. *P < 0.05, **P < 0.01, ***P < 0.001 between saline- and formalin-pretreated groups.

Figure 4

Autotomy onset and progression (A) and autotomy scores of individual digits (B) after medial injection prior to axotomy. Vertical marks indicate the mean onset and cutoff times in each group. ^#Lp < 0.05 vs. respective digits in lateral injection group.

Figure 5

Time course of PLP development as indicated by autotomy scores (A) and fraction of animals remaining (B) over time after medial injection prior to axotomy. *P < 0.05 between saline- and formalin-pretreated groups.

Figure 6

Autotomy onset and progression (A) and autotomy score of digits (B) after CCI prior axotomy. Vertical marks indicate the mean onset and cutoff times in each group. *P < 0.05, **P < 0.01 between indicated groups.

Figure 7

Time course of PLP development as indicated by autotomy scores (A) and fraction of animals remaining (B) over time after CCI (at 1 day or 4 weeks) prior to axotomy. *P < 0.05 for CCI 4w vs. Sham 4w (orange), ***P < 0.001 for both CCI vs. Sham (black) and CCI 1d vs. Sham 1d (red).

Figure 8

Correlation analysis of PLP scores post-axotomy and tactile hypersensitivity (tactile) and cold hypersensitivity (cold) after 1d (A,B) and 4 weeks (C,D) post-CCI, prior axotomy. R² and P-values for each analysis are indicated.

Figure 9

(A) Immunostaining of spinal dorsal horn for NaV1.7 in animals with severe and mild PLP behavior. (B) Evaluation of NaV1.7 total length in spinal cord from lumbar levels. (C) Correlation analysis between NaV 1.7 data and PLP scores. *P < 0.05 between the indicated groups.

Figure 10

(A) Immunostaining of spinal dorsal horn for Iba-1 in animals with severe and mild PLP behavior. (B) Evaluation of Iba-1 immunodensity in the lumbar spinal cord. (C) Correlation analysis of Iba-1 immunodensity and PLP scores. (D) Evaluation of IL-1β levels in CSF of animals with severe and mild PLP behavior. (E) Correlation analysis of IL-1β levels and PLP scores. *P < 0.05, **P < 0.01 between the indicated groups.

Figure 11

(A) Immunostaining of spinal dorsal horn for CGRP and GAD65/67 in animals with severe and mild PLP behavior. (B) Evaluation of CGRP immunodensity in the spinal laminae LI-III from lumbar levels. (C) Correlation analysis of CGRP Immunodensity and PLP scores. (D) Evaluation of GAD65/67 immunodensity in the lumbar spinal cord. (E) Correlation analysis of GAD65/67 immunodensity and PLP scores. *P < 0.05 between the indicated groups.