Spinal cord stimulation reduces cardiac pain through microglial deactivation in rats with chronic myocardial ischemia

Abstract

Angina pectoris is cardiac pain that is a common clinical symptom often resulting from myocardial ischemia. Spinal cord stimulation (SCS) is effective in treating refractory angina pectoris, but its underlying mechanisms have not been fully elucidated. The spinal dorsal horn is the first region of the central nervous system that receives nociceptive information; it is also the target of SCS. In the spinal cord, glial (astrocytes and microglia) activation is involved in the initiation and persistence of chronic pain. Thus, the present study investigated the possible cardiac pain‑relieving effects of SCS on spinal dorsal horn glia in chronic myocardial ischemia (CMI). CMI was established by left anterior descending artery ligation surgery, which induced significant spontaneous/ongoing cardiac pain behaviors, as measured using the open field test in rats. SCS effectively improved such behaviors as shown by open field and conditioned place preference tests in CMI model rats. SCS suppressed CMI‑induced spinal dorsal horn microglial activation, with downregulation of ionized calcium‑binding adaptor protein‑1 expression. Moreover, SCS inhibited CMI‑induced spinal expression of phosphorylated‑p38 MAPK, which was specifically colocalized with the spinal dorsal horn microglia rather than astrocytes and neurons. Furthermore, SCS could depress spinal neuroinflammation by suppressing CMI‑induced IL‑1β and TNF‑α release. Intrathecal administration of minocycline, a microglial inhibitor, alleviated the cardiac pain behaviors in CMI model rats. In addition, the injection of fractalkine (microglia‑activating factor) partially reversed the SCS‑produced analgesic effects on CMI‑induced cardiac pain. These results indicated that the therapeutic mechanism of SCS on CMI may occur partially through the inhibition of spinal microglial p38 MAPK pathway activation. The present study identified a novel mechanism underlying the SCS‑produced analgesic effects on chronic cardiac pain.

Keywords: cardiac pain; chronic myocardial ischemia; microglia; spinal cord stimulation.

Figure 1.

Experimental schedule. Timeline of SCS treatment, intrathecal implantation, drug administration and behavioral tests after CMI. (A) SCS was administered once a day from day 17 to 20 after CMI. Open field test and a morphological study were conducted at 21 days after CMI surgery. (B) Preconditioning and conditioning were performed from days 10 to 20, followed by CPP testing on day 21. (C) Mino and (D) Frac or vehicle were intrathecally administered daily from days 17 to 20 after CMI. The open field test was performed on day 21. CMI, chronic myocardial ischemia; CPP, conditioned place preference; frac, fractalkine; mino, minocycline; SCS, spinal cord stimulation; veh, vehicle.

Figure 2.

Representative images from Masson's trichrome staining in heart tissues from the different groups. Chronic infarct size in (A) naive, (B) sham and (C) CMI rats was determined using Masson's trichrome stain of heart tissue at the border zone of infarcted myocardium at cross sections of ventricular papillary muscles to label collagen scar tissue (blue) and cardiac muscle (red) 21 days after left anterior descending artery ligation surgery. An increased collagen area was observed in the CMI group. Scale bar, 100 µm. CMI, chronic myocardial ischemia.

Figure 3.

Behavioral assessments of rats in open field and conditioned place preference tests. (A) Vertical counts and (B) travel distance were decreased in rats with CMI in the open field test. SCS significantly increased the vertical counts but did not affect the distance traveled in CMI model rats. (C) Conditioning with SCS increased the time spent in the paired chamber of rats with CMI, but not in sham-operated rats, with a corresponding decrease in the non-SCS-paired chamber. N=8 rats/group; *P<0.05, ***P<0.001; ^##P<0.01 v. pre-conditioning values. NS, not significant; CMI, chronic myocardial ischemia; SCS, spinal cord stimulation.

Figure 4.

Changes in the immunoreactivity levels of GFAP and Iba-1 in the thoracic spinal cord after SCS in CMI model rats. Representative images of (A-C) GFAP-positive astrocytes and (D-F) Iba-1-positive microglia in the spinal dorsal horn in the different groups. Scale bar, 100 µm. Immunoreactivities for (G) GFAP and (H) Iba-1 within the dorsal horn were increased in the CMI group compared with those in the sham group. SCS effectively decreased the immunoreactivity for Iba-1 but not that of GFAP. N=8 rats/group; **P<0.01, ***P<0.001. CMI, chronic myocardial ischemia; GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor protein-1; ns, not significant; SCS, spinal cord stimulation.

Figure 5.

p-p38 MAPK is exclusively expressed in microglia in the spinal cord. Double-immunofluorescence staining showed that spinal p-p38 was not co-expressed with (A-D) GFAP-positive astrocytes or (E-H) NeuN-positive neurons. Instead, p-p38 was colocalized with (I-L) Iba-1-positive microglia. (D, H and L) Magnified images of the rectangles in (C, G and K, respectively). Arrows indicate typical double-labeled (yellow) cells and arrowhead indicates Iba-1 single-labeled microglia (red) in panel L. Scale bar, 100 µm in panels A-C, E-G and I-K; scale bar, 50 µm in panels D, H and L. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adaptor protein-1; NeuN, neuronal nuclei; p-, phosphorylated.

Figure 6.

P-p38 upregulation is inhibited by SCS in CMI model rats. Representative immunofluorescence staining images of spinal cord tissue showing the changes of p-p38 expression in (A) sham, (B) CMI and (C) CMI + SCS. Scale bar, 100 µm. (D) Number of p-p38-positive cells was greatly increased in CMI model rats and was partially inhibited by SCS. (E) Western blotting results were (F) semi-quantified, which indicated that the ratio of p-p38/p38 was elevated in CMI and inhibited by SCS. N=8 rats/group; *P<0.05, **P<0.01; ^#P<0.05 vs. sham. CMI, chronic myocardial ischemia; p-, phosphorylated; SCS, spinal cord stimulation.

Figure 7.

SCS inhibits CMI-induced upregulation of mRNA expression levels of pro-inflammatory mediators. Reverse transcription-quantitative PCR measurement showed that SCS significantly prevented CMI-induced increases in the levels of (A) IL-1β and (B) TNF-α in the spinal dorsal horn. N=8 rats/group; **P<0.01. CMI, chronic myocardial ischemia; SCS, spinal cord stimulation.

Figure 8.

Behavioral pharmacological assessments of CMI rats in open field tests. Microglial inhibition reduces CMI-induced cardiac pain, whereas microglial activation partially reverses the therapeutic effects of SCS on CMI model rats, as revealed by the open field test. The declined vertical count in CMI model rats was increased by i.t. Mino administration. This therapeutic effect was weaker compared with that of SCS treatment on CMI model rats. However, following i.t. Frac administration, the vertical count of CMI model rats after SCS was decreased. N=8 rats/group. *P<0.05, **P<0.01, ***P<0.001. CMI, chronic myocardial ischemia; Frac, fractalkine; i.t., intrathecal; Mino, minocycline; SCS, spinal cord stimulation; Veh, vehicle.