Spinal dorsal horn IGF1 mediates the preventive effect of electroacupuncture on cisplatin-induced peripheral neuropathy via neuronal IGF1R in mice

Abstract

Background: Our previous study demonstrated that neuronal G protein-coupled receptor kinase (GRK2) upregulation alleviated chemotherapy-induced peripheral neuropathy (CIPN) in mice, which was characterized by numbness and pain in distal hind limbs. The neuronal GRK2 was identified as a mediator of electroacupuncture (EA) effects on CIPN. Given that spinal insulin-like growth factor 1 (IGF1), a known inducer of GRK2 in the peripheral neurons, decreases after oxaliplatin treatment in mice, this study is designed to investigate whether spinal IGF1 contributes to EA-mediated prevention of cisplatin-induced peripheral neuropathy via neuronal IGF1 receptor (IGF1R).

Methods: A total of 133 male C57BL/6 J mice were included in this study and randomly assigned to different experimental groups. The level of Igf1 mRNA was detected by Real-time PCR, the p-IGF1R protein level by Western blot, after EA treatment in cisplatin-treated mice. The cellular distribution of p-IGF1R in the spinal dorsal horn was observed by immunofluorescent staining. To study the role of neuronal IGF1R in EA preventing cisplatin-induced mechanical allodynia, sensory deficit, and microglia activation and neuroinflammation in the spinal cord of mice, the neuronal IGF1R was downregulated by intraspinal injection of an AAV vector delivering IGF1R shRNA with hSyn promotor (AAV-shIGF1R). Finally, the regulatory effect of EA on spinal GRK2 was assessed by Western blot in AAV-shIGF1R mice.

Results: Cisplatin treatment induced mechanical allodynia, sensory deficit, and a decrease of p-IGF1R in the spinal dorsal horn of mice. Immunofluorescence showed that p-IGF1R was localized within neurons (~ 82%), a small mount of microglia (~ 12%) and astrocytes (~ 4%). Cisplatin decreased NeuN⁺p-IGF1R⁺ neurons in the spinal dorsal horn. EA treatment significantly alleviated cisplatin-induced mechanical allodynia, sensory deficit, and significantly increased the Igf1 mRNA and p-IGF1R level in the spinal cord. Neuronal IGF1R downregulation in the spinal dorsal horn significantly attenuated the preventive effect of EA on cisplatin-induced mechanical allodynia, sensory deficit, and spinal microglial activation and neuroinflammation in mice. Furthermore, neuronal IGF1R downregulation decreased the spinal GRK2 in cisplatin-treated mice after EA treatment. These findings suggest EA significantly alleviated CIPN symptoms by enhancing IGF1/IGF1R signaling and reducing microglial activation and neuroinflammation.

Conclusion: Spinal dorsal horn IGF1 contributes to the preventive effect of EA treatment against cisplatin-induced peripheral neuropathy through neuronal IGF1R signaling in mice. The enhanced neuronal IGF1/IGF1R signaling in the spinal cord presents a potential strategy for CIPN prevention.

Keywords: CIPN (Chemotherapy-induced peripheral neuropathy); Electroacupuncture; IGF1 (Insulin-like growth factor 1); Microglia activation; Neuroinflammation; Neuron.

Fig. 1

Cisplatin treatment induced mechanical allodynia and sensory deficit in mice. A Schedule of cisplatin treatment and behavioral test. B Repeated cisplatin treatments decreased the paw withdrawal threshold in von Frey test. Values are represented as mean ± SEM. n = 12 for saline group, 11 for cisplatin group. The statistical method is repeated measures ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, ***p < 0.001, η² = 0.24, 95% CI [0.24, 0.38]. C Repeated cisplatin treatments increased response time in adhesive removal test. Values are represented as mean ± SEM. n = 12 for saline group and cisplatin group. The statistical method is unpaired t-test. **p < 0.01, η² = 0.29, 95% CI [0.26, 1.40]. D Western blot analysis revealed a decrease of p-IGF1R, but no change of IGF1R in the spinal dorsal horn by downregulating neuronal IGF1R after cisplatin treatment. Results are normalized to β-actin. n = 8 for each group. Values are mean ± SEM. The statistical method is unpaired t-test. **p < 0.01, η² = 0.40, 95% CI [− 0.43, − 0.08]

Fig. 2

The cellular distribution of p-IGF1R in the spinal dorsal horn. A The representative images of p-IGF1R co-localized with NeuN in the spinal dorsal horn of saline- or cisplatin-treated mice. B, C The percentage of NeuN⁺p-IGF1R⁺ cells in p-IGF1R⁺ and NeuN⁺ cells. Values are represented as mean ± SEM. n = 4 for saline group and cisplatin group. The statistical method is unpaired t-test. *p < 0.01, η² = 0.59, 95% CI [− 51.1, − 5.14]. D The representative images of p-IGF1R co-localized with Iba1 in the spinal dorsal horn of saline- or cisplatin-treated mice. E–F The percentage of Iba1⁺p-IGF1R⁺ cells in p-IGF1R⁺ and Iba1⁺ cells. Values are represented as mean ± SEM. n = 4 for saline group and cisplatin group. The statistical method is unpaired t-test. G The representative images of p-IGF1R co-localized with GFAP in the spinal dorsal horn of saline- or cisplatin-treated mice. H–I The percentage of GFAP⁺p-IGF1R⁺ cells in p-IGF1R⁺ cells and GFAP⁺ cells. Values are represented as mean ± SEM. n = 4 for saline group and cisplatin group. The statistical method is unpaired t-test

Fig. 3

The effect of EA treatment on cisplatin-treated mice. A Schedule of cisplatin and EA treatment. B EA treatment increased the paw withdrawal threshold of cisplatin-treated mice in von Frey test. n = 8–9. Values are mean ± SEM. The statistical method is two-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, ***p < 0.001, η² = 0.22, 95% CI [0.21, 0.34]. C EA treatment decreased the response time of cisplatin-treated mice in adhesive removal test. n = 8–9. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, ***p < 0.001, η² = 0.5, 95% CI [0.30, 1.00]. D Real-time PCR analysis of Igf1 mRNA in the spinal dorsal horn of cisplatin-treated mice after EA treatment. n = 4 for cisplatin group, cisplatin + sham EA group and cisplatin + EA group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, η² = 0.59, 95% CI [− 1.59, − 0.06]. E The representative image of p-IGF1R and IGF1R protein in the spinal dorsal horn of cisplatin-treated mice after EA treatment. F–G The analysis of protein level of p-IGF1R and IGF1R in the spinal dorsal horn of cisplatin-treated mice after EA treatment. Results are normalized to β-actin and shown as ratios to cisplatin-treated mice. n = 5 for cisplatin group, cisplatin + sham EA group and cisplatin + EA group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, η² = 0.44, 95% CI [− 0.80, − 0.04]

Fig. 4

Downregulation of neuronal IGF1R in the spinal dorsal horn after intraspinal injection of adeno-associated virus. A The working mode of neuronal IGF1R downregulated through intraspinal injection of adeno-associated virus. B The immunofluorescent results of AAV-expressed eGFP (green) co-localizes with NeuN (red) in the spinal dorsal horn. White arrow: representative neuron (red) expressed eGFP (green). Scale bar: 100 μm. C Western blot analysis revealed a decrease of IGF1R in the spinal dorsal horn after intraspinal injection of adeno-associated virus. Results are normalized to β-actin. n = 4 for each group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, η² = 0.62, 95% CI [0.08, 0.57]. D–E Effect of neuronal IGF1R downregulation on locomotor performance in open field test and rotarod test. n = 8 for each group. Values are mean ± SEM. The statistical method is one-way ANOVA

Fig. 5

Downregulation of neuronal IGF1R in the spinal dorsal horn inhibited the preventive effect of EA on cisplatin-induced mechanical allodynia and sensory deficits. A Schedule of neuronal IGF1R knockdown and EA treatment in cisplatin-treated mice. Baseline was defined as day 0, corresponding to the time point after the final tamoxifen injection when the viral construct was activated. Electroacupuncture treatment was initiated after baseline behavioral test, one day prior to cisplatin injection. B Downregulation of neuronal IGF1R decreased paw withdrawal threshold following EA treatment. n = 10–12. Values are mean ± SEM. The statistical method is two-way repeated measures ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, ***p < 0.001, η² = 0.93, 95% CI [0.22, 0.43] C Downregulation of neuronal IGF1R extended the response time in mice following EA treatment. n = 10–11. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, ***p < 0.001, η² = 0.47, 95% CI [− 1.41, 0.34]

Fig. 6

Downregulation of neuronal IGF1R in the spinal dorsal horn inhibited the preventive effect of EA on spinal microglia activation and neuroinflammation. A Immunofluorescence staining of Iba1 in cisplatin-treated mice after virus administration and EA treatment. Scale bars: 100 μm. B The analysis of Iba1 immunoreactivity. n = 4 for each group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, ***p < 0.001, η² = 0.86, 95% CI [− 6.25, − 2.27]. C–J Real-time PCR analysis of Cd16, Il-1β, Il-6, Tnf-α, inos, Il-4, Il-10, Cd206. Results are normalized to Gapdh. n = 6 for each group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, **p < 0.01, Cd16:η² = 0.43, 95% CI [− 1.81, − 0.07]; Il-1β: η² = 0.61, 95% CI [− 1.96, − 0.42]; Il-6: η² = 0.57, 95% CI [− 1.07, − 0.24]; Tnf-α::η² = 0.35, 95% CI [− 2.11, − 0.02]

Fig. 7

Western blot analysis revealed a decrease of GRK2 in the spinal dorsal horn by downregulating neuronal IGF1R after electroacupuncture treatment. Results are normalized to β-actin. n = 6 for each group. Values are mean ± SEM. The statistical method is one-way ANOVA followed by Tukey test for multiple comparisons. For post hoc analysis, *p < 0.05, η² = 0.41, 95% CI [0.04, 0.47]

Fig. 8

Schematic representation of our working model. EA upregulated IGF1, which regulated neuronal GRK2 through IGF1R, and then microglia activation and neuroinflammation in the spinal dorsal horn, finally contributing to the preventive process on cisplatin-induced CIPN by EA. This preventive process was inhibited by silencing the neuronal IGF1R in cisplatin-treated mice