Cerebrospinal fluid-contacting nucleus mediates nociception via release of fractalkine

Increasing evidence suggests that the cerebrospinal ﬂuid-contacting nucleus (CSF-contacting nucleus) mediates the transduction and regulation of pain signals. However, the precise molecular mechanisms remain unclear. Studies show that release of fractalkine (FKN) from neurons plays a critical role in nerve injury-related pain. We tested the hypothesis that release of FKN from the CSF-contacting nucleus regulates neuropathic pain, in a chronic constriction injury rat model. The results show that FKN is expressed by neurons, via expression of its only receptor CX3CR1 in the microglia. The levels of soluble FKN (sFKN) were markedly upregulated along with the increase in FKN mRNA level in rats subjected to chronic constriction injury. In addition, injection of FKN-neutralizing antibody into the lateral ventricle alleviated neuropathic pain-related behavior followed by reduction in microglial activation in the CSF-contacting nucleus. The results indicate that inhibition of FKN release by the CSF-contacting nucleus may ameliorate neuropathic pain clinically.

Cerebrospinal fluid-contacting nucleus; FKN; microglia; Neuropathic pain; Chronic constriction injury

Authorship

Q.Q. Zhou ^**These authors contributed equally to this study.

Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu Province, ChinaXuzhou Medical UniversityChinaXuzhou, Jiangsu Province, ChinaJiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou, Jiangsu Province, China

Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu Province, ChinaXuzhou Medical UniversityChinaXuzhou, Jiangsu Province, ChinaJiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou Medical University, Xuzhou, Jiangsu Province, China

S.S. Chen ^**These authors contributed equally to this study.

Q.Q. Zhang ^**These authors contributed equally to this study.

P.F. Liu

H.Z. Fang

Y. Yang

L.C. Zhang Correspondence: L.C. Zhang: <licaizhang001@163.com>

Correspondence: L.C. Zhang: <licaizhang001@163.com>

*These authors contributed equally to this study.

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Figures

Figures (6)

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Figure 1.
Behavioral testing of chronic constriction injury (CCI) rats. A, Duration of thermal withdrawal latency (F=61.487); B, paw withdrawal threshold (F=53.652). **P<0.01 compared with sham group at the same time point; ^##P<0.01 compared with baseline, n=7 per group (ANOVA with repeated measurements).

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Figure 2.
Expression of fractalkine (FKN) and CX3CR1 in the CSF-contacting nucleus. A-C, Cholera toxin subunit B-labeled positive neurons (red) and FKN-immunoreactive neurons (green) were co-expressed in specific neurons (yellow in merged image). D-F, Colocalization of Iba-1 (red) and CX3CR1 (green) immunoreactivity (yellow in merged image). n=3 per group, Scale bar =100 μm. Aq: aqueduct. CSF: cerebrospinal fluid.

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Figure 3.
Fractalkine (FKN) mRNA expression was increased after chronic constriction injury (CCI) surgery. Real-time PCR was conducted with total RNA samples extracted from the CSF-contacting nucleus in naive, sham, and CCI rats on days 3, 7, 14, and 21. **P<0.01 vs sham group, n=5, F=48.2 (one-way ANOVA, LSD test). CSF: cerebrospinal fluid.

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Figure 4.
Western blot of soluble fractalkine (sFKN) expression. Protein samples were isolated from the CSF-contacting nucleus in naive, sham, and chronic constriction injury (CCI) rats on days 3, 7, 14, and 21. *P<0.05 vs sham group, **P<0.01 vs sham group, n=5, F=19.065 (one-way ANOVA, LSD test). CSF: cerebrospinal fluid.

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Figure 5.
Fractalkine (FKN)-neutralizing antibody prevented chronic constriction injury (CCI)-induced pain. A, Thermal hyperalgesia after administration (F=58.575). B, Paw withdrawal threshold (F=92.935). *P<0.05 vs CCI + IgG group, ^#P<0.05 vs Sham + Anti-FKN group, n=8 per group (two-way ANOVA, LSD test).

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Figure 6.
Fractalkine (FKN)-neutralizing antibody attenuated the microglial response following chronic constriction injury (CCI). CB-labeled neurons (green in A-D) were used to locate the CSF-contacting nucleus. The cells testing positive for Iba-1 (red in E-H) within the box in the areas to the left and the right of CSF-contacting nucleus were counted. I, Quantification of Iba-1 immunoreactivity. **P<0.01 vs the CCI + IgG group, n=5 per group, F=240.048 (two-way ANOVA, LSD test). Scale bar =100 μm. CSF: cerebrospinal fluid.

Figure 1. Behavioral testing of chronic constriction injury (CCI) rats. A, Duration of thermal withdrawal latency (F=61.487); B, paw withdrawal threshold (F=53.652). **P<0.01 compared with sham group at the same time point; ^##P<0.01 compared with baseline, n=7 per group (ANOVA with repeated measurements).

Figure 2. Expression of fractalkine (FKN) and CX3CR1 in the CSF-contacting nucleus. A-C, Cholera toxin subunit B-labeled positive neurons (red) and FKN-immunoreactive neurons (green) were co-expressed in specific neurons (yellow in merged image). D-F, Colocalization of Iba-1 (red) and CX3CR1 (green) immunoreactivity (yellow in merged image). n=3 per group, Scale bar =100 μm. Aq: aqueduct. CSF: cerebrospinal fluid.

Figure 3. Fractalkine (FKN) mRNA expression was increased after chronic constriction injury (CCI) surgery. Real-time PCR was conducted with total RNA samples extracted from the CSF-contacting nucleus in naive, sham, and CCI rats on days 3, 7, 14, and 21. **P<0.01 vs sham group, n=5, F=48.2 (one-way ANOVA, LSD test). CSF: cerebrospinal fluid.

Figure 4. Western blot of soluble fractalkine (sFKN) expression. Protein samples were isolated from the CSF-contacting nucleus in naive, sham, and chronic constriction injury (CCI) rats on days 3, 7, 14, and 21. *P<0.05 vs sham group, **P<0.01 vs sham group, n=5, F=19.065 (one-way ANOVA, LSD test). CSF: cerebrospinal fluid.

Figure 5. Fractalkine (FKN)-neutralizing antibody prevented chronic constriction injury (CCI)-induced pain. A, Thermal hyperalgesia after administration (F=58.575). B, Paw withdrawal threshold (F=92.935). *P<0.05 vs CCI + IgG group, ^#P<0.05 vs Sham + Anti-FKN group, n=8 per group (two-way ANOVA, LSD test).

Figure 6. Fractalkine (FKN)-neutralizing antibody attenuated the microglial response following chronic constriction injury (CCI). CB-labeled neurons (green in A-D) were used to locate the CSF-contacting nucleus. The cells testing positive for Iba-1 (red in E-H) within the box in the areas to the left and the right of CSF-contacting nucleus were counted. I, Quantification of Iba-1 immunoreactivity. **P<0.01 vs the CCI + IgG group, n=5 per group, F=240.048 (two-way ANOVA, LSD test). Scale bar =100 μm. CSF: cerebrospinal fluid.

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Cerebrospinal fluid-contacting nucleus mediates nociception via release of fractalkine

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[1] Correspondence: L.C. Zhang: <licaizhang001@163.com>