Pretreatment with intrathecal amitriptyline potentiates anti-hyperalgesic effects of post-injury intra-peritoneal amitriptyline following spinal nerve ligation

Abstract

Background: Amitriptyline, a tricyclic antidepressant and potent use-dependent blocker of sodium channels, has been shown to attenuate acute and chronic pain in several preclinical modes. The purpose of this study was to investigate whether intrathecal pretreatment with amitriptyline combined with post-injury intra-peritoneal amitriptyline is more effective than post-injury treatment alone on L5 spinal nerve ligation (SNL)-induced neuropathic pain.

Methods: 96 adult male Sprague-Dawley rats were allocated into 4 groups: group S, Sham; group L, L5 spinal nerve Ligation with vehicle treatment; group A, SNL and post-injury intra-peritoneal (Abdominal) amitriptyline twice daily × 3 days; group P, intrathecal Pretreatment with amitriptyline, SNL and intra-peritoneal amitriptyline twice daily × 3 days. Responses to thermal and mechanical stimuli, as well as sodium channel expression in injured dorsal root ganglion (DRG) and activated glial cells in spinal dorsal horn (SDH) were measured pre-operatively and on post-operative day (POD) 4, 7, 14, 21 and 28.

Results: SNL-evoked hyper-sensitivity responses to thermal and mechanical stimuli, up-regulated Nav1.3 and down-regulated Nav1.8 expression in DRG, and activated microglia and astrocytes in SDH. In group A, intra-peritoneal amitriptyline alone alleviated thermal hypersensitivity on POD7, reversed Nav1.8 and reduced activated microglia on POD14. In group P, intrathecal pretreatment with amitriptyline not only potentiated the effect of intra-peritoneal amitriptyline on thermal hypersensitivity and Nav1.8, but attenuated mechanical hypersensitivity on POD7 and reduced up-regulated Nav1.3 on POD14. Furthermore, this treatment regimen reduced astrocyte activation on POD14.

Conclusions: Concomitant intrathecal pretreatment and post-injury intra-peritoneal amitriptyline was more effective than post-injury treatment alone on attenuation of behavioral hypersensitivity, decrease of activated microglia and astrocytes and dysregulated Nav1.3 and 1.8.

Figure 1

Flow chart of the study. DRG: dorsal root ganglion; D: day; m: minute; POD: postoperative day; S: Sham; L: Ligation followed by vehicle treatment; A: ligation followed by treatment with amitriptyline intra-peritoneally post-injury; P: Pretreatment intrathecal amitriptyline, ligation and post-injury intra-peritoneal amitriptyline.

Figure 2

Differences in withdrawal latency between left and right hind paws after thermal (A) and mechanical (B) stimulation. There are no significant differences in withdrawal latencies between hindpaws in the sham group (S). Spinal nerve ligation (group L) produced a significant difference in withdrawal latencies between hindpaws. Intra-peritoneal amitriptyline post-injury (group A) alleviated thermal hypersensitivity for one week. Pretreatment intrathecal amitriptyline in combination with post-injury intra-peritoneal amitriptyline (group P) alleviated thermal hypersensitivity for 2 weeks and attenuated mechanical hypersensitivity for 1 week. POD: postoperative day, (red accent diamond: group S, red down-pointing triangle: group L, green circle: group A, olive green square: group P, n = 24/group, 4-5/each time point). L vs. S, ☆: P < 0.001; A vs. L, c: P < 0.001; P vs. L,#: P < 0.001. Group differences were compared by ANOVA followed by Scheffe test of multiple post hoc analyses. Error bars represent SE.

Figure 3

Protein expressions of Nav1.3, Nav1.8, and Nav1.7 in left L5 DRG by western blot analyses. Quantification of Nav levels at each time point for each group was normalized against the Nav levels of the sham group. SNL (group L) induced up-regulation of Nav1.3 (A) and down-regulated Nav1.8 (B) for 28 days. Treatment with amitriptyline intra-peritoneally (group A) did not have a significant effect on SNL-induced up-regulation of Nav1.3. Pretreatment with intrathecal amitriptyline together with post-injury treatment with the same compound intra-peritoneally (group P) decreased SNL-induced up-regulation of Nav1.3 for 2 weeks (A). Significant inhibition of SNL-induced down-regulated Nav1.8 was found in groups A and P; the effect lasting for 2 and 3 weeks, respectively (B). Furthermore, both amitriptyline regimens reversed the SNL-induced down-regulation of Nav1.7 on POD4 (C)_. Data represent mean ± SE. One way ANOVA (n = 24/group, 4-5/each time point). *p < 0.05; **p < 0.01; ☆:p < 0.001,NS: not significant. POD: postoperative day; S: Sham; L: Ligation followed by vehicle treatment; A: ligation and treatment with amitriptyline intra-peritoneally (Abdomen) post-injury; P: Pretreatment with intrathecal amitriptyline, ligation and intra-peritoneal amitriptyline post-injury.

Figure 4

Immunofluorescence staining (A) and quantification of the expression of OX-42 (B) in microglia of the spinal dorsal horn (SDH). SNL (group L) induced a dramatic increase in OX-42 positive microglia in the SDH. Both amitriptyline regimens (group A and P) reduced the expression of OX-42 immunoreactivity in microglia for 2 weeks (B). Group P showed a greater effect than group A on reduction of activated microglia in SDH on POD4. Scale bars = 200 μm. Data represent mean ± SE, the Mann–Whitney U test was used for statistical comparison. (n = 24/group, 4-5/each time point). *p < 0.05; **p < 0.01, NS: not significant. POD: postoperative day; S: Sham; L: Ligation; A: post-injury intra-peritoneal amitriptyline; P: Pretreatment with intrathecal amitriptyline in addition to post-injury intra-peritoneal amitriptyline.

Figure 5

Immunofluorescence staining (A) and quantification of the expression of GFAP (B) in astrocytes of the spinal dorsal horn (SDH). Low immunoreactivity was observed in the sham rats (group S). SNL (group L) increased the immunoreactivity of GFAP-positive astrocytes in the SDH. Intra-peritoneal treatment with amitriptyline (group A) had no effect on decreasing the SNL-induced activation of astrocytes. Intrathecal pretreatment in combination with intra-peritoneal administration of amitriptyline (group P) decreased the activation of astrocytes for 2 weeks (group P vs. L). Scale bars = 200 μm. Data represent mean ± SE, the Mann–Whitney U test was used for statistical comparison. (n = 24/group, 4-5/each time point). *p < 0.05, ns: not significant. POD: postoperative day; S: Sham; L: Ligation; A: post-injury intra-peritoneal amitriptyline; P: Pretreatment with intrathecal amitriptyline in addition to post-injury intra-peritoneal amitriptyline.