ABSTRACT
BACKGROUND AND OBJECTIVES:
Half of neuropathic pain patients still end up failing clinical treatments. Electrical stimulation of the posterior insular cortex (ESI) modulates sensory and nociceptive circuits. This study evaluated the effects of a range of frequencies of ESI proposed to improve neuropathic pain.
METHODS:
Male Sprague Dawley rats, 280-340 g, submitted to the chronic constriction of the right sciatic nerve were tested for mechanical sensitivity using the paw pressure and von Frey flaments tests, and for thermal sensitivity using the hot plate test. The rats were submitted to ESI 10, 60 or 100 Hz (one, five or seven ESI, 15 min, 210 µs, 1V), applied to the posterior insular cortex, and were evaluated in the tests before and after ESI, or in follow-up of 48, 72 and 168h. The open field evaluated general activity after ESI 5. The involvment of opioid and cannabinoid testes were evaluated through treatment with naloxone and SR1416A - antagonist and inverse agonist/antagonist of the receptors, respectively, after ESI 5, while activation of astrocytes, marked by glial fibrillary acid protein (GFAP), and of microglia, marked by IBA-1 (glial marker), in the spinal cord evaluated by immunohistochemistry.
RESULTS:
Data demonstrate that 10, 60, and 100 Hz ESIs modulate mechanical and thermal sensitivity. ESI 5 increased immunoreactivity of GFAP in the spinal cord, without altering IBA-1 (glial marker). Naloxone and SR141716A reversed the antinociception of 60 Hz ESI 5. 60 Hz ESI 7 induced antinociception up to 72h.
CONCLUSION:
60 Hz ESI induces opioid and cannabinoid-dependent antinociception and regulates glia.
HIGHLIGHTS
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60 Hz-delivered ESI was the best analgesic protocol for the insular stimulation.
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Data showed a prolonged analgesic effect up to 72h after repetitive ESI.
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ESI regulates glia activation in pain modulatory system.
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CCI-rats with electrodes implanted in left posterior insular cortex were submitted to repetitive ESI delivered at 10, 60, and 100Hz and evaluated in the paw pressure test (A to C) and using von Frey flaments (D to F) at baseline, before (pre) and after (post) a single or five ESI sessions. Groups were separated in (A) ESI 10Hz (n = 8), (B) ESI 60Hz (n = 8), (C) ESI 100Hz (n = 5); and SHAM (n = 4 to 5). Data is presented as mean ± S.E.M. Two-way ANOVA was followed by the Bonferroni post-test. (A) baseline vs Pre ESI 1 1, ****p <0.0001. (B) Baseline vs Pre ESI 1; Pre ESI 1 vs Post ESI 1; Pre ESI 1 vs Pre ESI 5; Pre ESI 1 vs Post ESI 5, ****p <0.0001. Pre ESI 5 vs Post ESI 5; **p = 0.0048; Groups SHAM vs ESI, Post ESI 1 and Post ESI 5 ####p<0.0001. (C) Baseline vs Pre ESI 1, ****p <0.0001; Pre ESI 1 vs Post ESI 1 and Post ESI 5, ****p <0.0001; Pre ESI 5 vs Post ESI 5; ****p <0.0001; Groups SHAM vs ESI, Post ESI 1 and ESI 5 ####p<0.0001; (D ) Baseline vs Pre ESI 1, ****p< 0.0001; Pre ESI 1 vs Post ESI 1, *p = 0.0211; Pre ESI 1 vs Pre ESI 5, **p = 0.0015; Pre ESI 1 vs Post ESI 5, ****p< 0.0001. Groups SHAM vs ESI, Post ESI 1, Pre ESI 5; Post ESI 5 ##p= 0.0013; #p= 0.0227; ####p<0.0001, respectively. (E) Baseline vs Pre ESI 1, ****p<0.0001; Pre ESI 1 vs Post ESI 1, **p = 0.0244; Pre ESI 1 vs Post ESI 5, ****p<0.0001. Groups SHAM vs ESI, Post ESI 1, Post ESI 5 ####p<0.0001. (F) Baseline vs Pre ESI 1; ****p< 0.0001; Pre ESI 1 vs Post ESI 1, **** p<0.0001; Groups SHAM vs ESI, Post ESI 1, Pre ESI 5, Post ESI 5 ####p<0.0001.
CCI-rats implanted with electrodes in the left posterior insular cortex were submitted to repetitive ESI delivered at 10, 60, 80 and 100 Hz and evaluated in hot plate test for thermal hypersensitivity at baseline, before (pre) and after (post) a single or five ESI sessions. For thermal sensitivity analyses, groups were separated in (A) ESI 10 Hz (n = 5), (B) ESI 60 Hz (n = 4), (C) ESI 100 Hz (n = 4); and SHAM (n = 4). General activity was measured in the open field test once after five ESI. Raising (D) and locomotion (E) were assessed in 10 Hz ESI (n=3), 60 Hz ESI (n=3), 100 Hz ESI (n=3) and SHAM (n=3) animals during three minutes. Data presented as mean ± S.E.M. In (A-C), two-way ANOVA followed by the Bonferroni post-hoc test. In (A) Baseline vs Pre ESI 1, ****p< 0.0001. (B) Pre ESI 1 vs Post ESI 1, **p=0.0066; Baseline vs Pre ESI 1, Pre ESI 1 vs Pre ESI 5 and Pre ESI 1 vs Post ESI5, ****p< 0.0001. Groups SHAM vs ESI, Post ESI 1, ##p=0.0076; Post ESI 5, ####p<0.0001. In (C) Baseline vs Pre ESI 1; ****p< 0.0001. (D-E), one-way ANOVA followed by the Bonferroni post-hoc test.
CCI-rats implanted with electrodes in the left posterior insular cortex submitted to repetitive 60Hz ESI were injected with (A) naloxone (2 mg/kg; i.p.) (ESI+Sal n= 6; ESI+NLX n=5) or (B) SR141716 (1 mg/kg; i.p.) (ESI+DMSO n= 4; ESI+ SR141716 n=4) 30 min and 40 min prior to mechanical nociceptive evaluation, respectively. Animals were evaluated in paw pressure test in a baseline, before (pre) and after (post) a single or five 60Hz ESI sessions. Data presented as mean ± S.E.M. Two-way ANOVA followed by the Bonferroni post-hoc test. (A) 60 HZ ESI +Sal vs 60 HZ ESI +NLX ***p=0.0006 (B) ESI 60 HZ +DMSO vs 60 HZ ESI +SR141716 ****p<0.0001. Abbreviations: Dimethyl sulfoxide (DMSO).
Fixed spinal cord slices of 60Hz ESI (n = 5) or SHAM (n = 4-5) CCI-rats were prepared for GFAP (C-F) or IBA-1 (H-K) immunohistochemistry. Representative images of the DHSC were obtained using an optical microscope, and optical density immunoreactivity analyzed by a threshold-based counting in Image J software. In A and B, horizontal bar = 100 μm; in all others, horizontal bar = 20 μm. GFAP-IR is marked by dark black points in the image and pointed by black arrows. In (H) and (L), data is presented as mean ± S.E.M. and was analyzed by two-way ANOVA followed by the Bonferroni post-hoc test. In (E) GFAP optical density SHAM vs ESI group in the right DHSC, * p = 0.0024.
CCI-rats implanted with electrodes in the left posterior insula were submitted to seven daily repetitive 60 Hz ESI (n=7) and evaluated in the paw pressure test at baseline, before (pre) and after (post) a single ESI (ESI 1), seven ESI sessions (ESI 7) and after different follow up periods (48h, 72h, 168h). Data is presented as mean ± S.E.M. One ANOVA was performed after the Bonferroni post-test. Baseline vs Pre ESI 1, ****p< 0.0001; Pre ESI 1 vs Post ESI 1, **p=0.0015; Pre ESI 1 vs Post ESI 7, *p=0.0465; Post ESI 7 vs 168h, **p=0.0030.