C3/C3aR Bridges Spinal Astrocyte-Microglia Crosstalk and Accelerates Neuroinflammation in Morphine-Tolerant Rats

Abstract

Aims: Communication within glial cells acts as a pivotal intermediary factor in modulating neuroimmune pathology. Meanwhile, an increasing awareness has emerged regarding the detrimental role of glial cells and neuroinflammation in morphine tolerance (MT). This study investigated the influence of crosstalk between astrocyte and microglia on the evolution of morphine tolerance.

Methods: Sprague-Dawley rats were intrathecally treated with morphine twice daily for 9 days to establish morphine-tolerant rat model. Tail-flick latency test was performed to identify the analgesic effect of morphine. The role of microglia, astrocyte and C3-C3aR axis in morphine tolerance were elucidated by real-time quantitative polymerase chain reaction, Western blot, and immunofluorescence.

Results: Chronic morphine treatment notably promoted the activation of microglia, upregulated the production of proinflammatory mediators (interleukin-1 alpha (IL-1α), tumor necrosis factor alpha (TNFα), and complement component 1q (C1q)). Simultaneously, it programed astrocytes to a pro-inflammatory phenotype (A1), which mainly expresses complement 3 (C3) and serping1. PLX3397 (a colony-stimulating factor 1 receptor (CSF1R) inhibitor), Compstain (a C3 inhibitor) and SB290157(a C3aR antagonist) could reverse the above pathological process and alleviate morphine tolerance to different extents.

Conclusion: Our findings identify C3-C3aR axis as an amplifier of microglia-astrocyte crosstalk, neuroinflammation and a node for therapeutic intervention in morphine tolerance.

Keywords: A1 astrocyte; C3/C3aR; microglia‐astrocyte crosstalk; morphine tolerance.

FIGURE 1

Experimental schematic diagram. (A) Experimental protocol and time course. (B) Animal experimental groups. The number of rats used for WB, qRT‐PCR, and IF was 5, 5, and 3, respectively. Compstain, a C3 inhibitor; i.t., intrathecal injection; IF, immunofluorescence; n, number of animals; PLX3397, a CSF1R inhibitor; qRT‐PCR, real‐time quantitative polymerase chain reaction; SB290157, a C3aR antagonist; Vehicle, 10% DMSO, 5% Tween 80 and 85% saline; WB, Western blot.

FIGURE 2

PLX3397 reduced microglia activation and alleviated A1 astrocyte activation in morphine‐tolerant rats. The PLX3397 (20 μg/5 μL) was intrathecally injected 30 min before morphine administration twice daily for 9 days. (A–D) The results of western blot showed that pretreatment with PLX3397 reduced protein expressions of Iba1, C1qA, ILL‐1α, TNF‐α induced by chronic morphine treatment (#p < 0.05, ##p < 0.01, ###p < 0.001 vs. Morphine + Vehicle group, *p < 0.05, ***p < 0.001, ****p < 0.0001 vs. NS + Vehicle group, n = 5 per group). (E, F) The results of immunofluorescence staining showed that pretreatment with PLX3397 reduced the number of Iba‐1 positive cells in morphine‐tolerant rats (Scale bar: 100 μm) (##p < 0.01 vs. Morphine + Vehicle group, ***p < 0.001 vs. NS + Vehicle group, n = 3 per group, three sections per sample were measured.). (G, H) The results of western blot showed that pretreatment with PLX3397 reduced protein expressions of C3 and SERPING1 induced by chronic morphine treatment (##p < 0.01, ###p < 0.001 vs. Morphine + Vehicle group, ***p < 0.001 vs. NS + Vehicle group, n = 5 per group). (I) The %MPE in rats receiving PLX3397 30 min before morphine administration were significantly higher than those in morphine‐tolerant rats from Day 7 to 9 (###p < 0.001, ####p < 0.0001 vs. Morphine + Vehicle group, ****p < 0.0001 vs. NS + Vehicle group, n = 5 per group). n, number of animals; NS, normal saline; Vehicle, 10% DMSO, 5% Tween 80 and 85% saline.

FIGURE 3

Compstain alleviated A1 astrocyte activation and reduced microglia activation in morphine‐tolerant rats. C3 inhibitor compstain (10 μg/5 μL) was intrathecally injected 30 min before morphine administration twice daily for 9 days. (A) The %MPE in rats receiving compstain 30 min before morphine administration were higher than those in morphine‐tolerant rats from Day 7 to 9 (###p < 0.001, ####p < 0.0001 vs. Morphine group, *p < 0.05, ****p < 0.0001 vs. NS group, n = 5 per group). (B, C) Compstain decreased the protein level of C3 and SERPING1 induced by chronic treatment of morphine (#p < 0.05, ##p < 0.01 vs. Morphine group, **p < 0.01, ***p < 0.001 vs. NS group, n = 5 per group). (D–G) Compstain decreased the protein level of Iba1, C1qA, TNF‐α and IL‐1α induced by chronic morphine treatment (##p < 0.01, ###p < 0.001, ####p < 0.0001 vs. Morphine group, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. NS group, n = 5 per group). n, number of animals; NS, normal saline. (H) The results of immunofluorescence staining showed that pretreatment with compstain reduced the expression of C3 and SERPING1 in the spinal cord of morphine‐tolerant rats (Scale bar: 100 μm) (##p < 0.01, ###p < 0.001 vs. Morphine group, **p < 0.01, ***p < 0.001 vs. NS group, n = 3 per group, three sections per sample were measured).

FIGURE 4

Cellular localization of C3 in the spinal dorsal horn of morphine‐tolerant rats. The result of dual‐label immunofluorescence showed that C3 was colocalized with NeuN, some with GFAP and Iba1 in the spinal dorsal horn of morphine‐tolerant rats (Scale bar: 100 μm, n = 3 per group, three sections per sample were measured). MT, morphine treatment; n, number of animals; NS, normal saline.

FIGURE 5

Chronic morphine treatment increased the expression of C3aR. (A, B) The results of qRT‐PCR and Western blot showed that chronic morphine treatment increased the expression of C3aR (**p < 0.01, ****p < 0.0001 vs. NS group, n = 5 per group). (C) The results of dual‐label immunofluorescence showed that C3aR was colocalized with NeuN, some with GFAP and Iba1 in the spinal dorsal horn of morphine‐tolerant rats (Scale bar: 100 μm, n = 3 per group, three sections per sample were measured). MT, morphine treatment; n = number of animals; NS, normal saline.

FIGURE 6

SB290157 alleviated A1 astrocyte and microglia activation and attenuated morphine tolerance (MT). C3aR inhibitor SB290157 (10 μg/5 μL) was intrathecally injected 30 min before morphine administration twice daily for 9 days. (A) Pretreatment with SB290157 attenuated the development of MT (####p < 0.0001 vs. Morphine + Vehicle group, ***p < 0.001, ****p < 0.0001 vs. NS + Vehicle group, n = 5 per group). (B, C) Pretreatment with SB290157 reduced the increased protein level of C3 and SERPING1 induced by chronic treatment of morphine (#p < 0.05, ##p < 0.01 vs. Morphine + Vehicle group, **p < 0.01 vs. NS + Vehicle group, n = 5 per group). (D–G) Pretreatment with SB290157 reduced the increased protein level of Iba1, C1qA, TNF‐α and IL‐1α induced by chronic morphine treatment (#p < 0.05, ##p < 0.01, ####p < 0.0001 vs. Morphine + Vehicle group, *p < 0.05, **p < 0.01, ****p < 0.0001 vs. NS + Vehicle group, n = 5 per group). n, number of animals; NS, normal saline; Vehicle, 10% DMSO, 5% Tween 80 and 85% saline. (H) The results of immunofluorescence staining showed that pretreatment with SB290157 reduced the expression of C3 and SERPING1 in the spinal cord of morphine‐tolerant rats (Scale bar: 100 μm, ##p < 0.01 vs. Morphine+ Vehicle group, **p < 0.01, ***p < 0.001 vs. NS + Vehicle group, n = 3 per group, three sections per sample were measured).