损伤与未损伤的初级感觉神经元和外周纤维中Cav3.2 T型钙离子通道参与神经病理痛的分子机制

Cav3.2 T-type calcium channels are predominant subtypes in peripheral pain transmission system. These channels participate in neuronal afterdepolarizing potentials and membrane oscillations, thus play a crucial role in neuronal excitability. So, the function of these channels in neuropathic pain got more attention recently. However, in different neuropathic pain models, there existed apparent contradictions about Cav3.2 T-type calcium channel current and expression, and behavioral pharmacology results. Based on other reports and our preliminary data, we found that the possible reason for these contradictions may be that previous studies did not distinguish injured sensory neurons and nerve fibers from the uninjured ones. The present project proposes that Cav3.2 T-type calcium channels in injured and uninjured DRG neurons and nerve fibers contribute to neuropathic pain differentially through participation in ectopic discharges or spontaneous discharges. To address this issue, we will investigate the role of Cav3.2 T-type calcium channels in neuropathic pain rats in the spared nerve injury (SNI) model with multiple-level methods, including whole-cell patch clamp, electrophysiological recording of single fiber firings, Western blot, immunofluorescent staining and behavioral tests. The first scientific question in the present project is whether the electrophysiological property, expression pattern and distribution of Cav3.2 T-type calcium channel are changed, and these changes contribute to neuropathic pain through ectopic discharges in injured and spontaneous discharges in uninjured DRG neurons and fibers. The second scientific question is whether Cav3.2 T-type calcium channels are redistributed to injured and uninjured nerve fibers, and these redistributions contribute to neuropathic pain through ectopic discharges and spontaneous discharges. This project will prove peripheral Cav3.2 T-type calcium channels contribute to neuropathic pain, and raise a possibility of targeting peripheral Cav3.2 T-type calcium channels for the treatment of neuropathic pain.

在痛觉外周传导通路中，Cav3.2 T型钙离子通道占主导，它参与神经元后去极化电位及膜电位振荡，影响神经元兴奋性。因此，Cav3.2 T型钙离子通道参与神经病理痛的机制受到重视。但是在不同神经病理痛模型，Cav3.2 T型钙离子通道电流和含量及行为药理学结果等相互矛盾。分析文献并结合预实验结果，我们发现，出现上述矛盾的原因可能在于：已有研究没有区分损伤与未损伤的背根神经节(DRG)神经元和外周纤维。据此，本项目将采用坐骨神经分支选择性损伤(SNI)模型，以损伤或未损伤的DRG神经元及外周纤维为研究对象，采用全细胞膜片钳技术、神经单纤维放电记录技术、分子生物学并结合行为药理学等方法，以期阐明Cav3.2 T型钙离子通道参与损伤与未损伤DRG和外周纤维的异位放电或自发放电，进而参与神经病理痛的分子机制。该项目也为钙离子通道特异性阻断剂用于神经病理痛的治疗提供启示。

背景：在痛觉外周传导通路中，Cav3.2 T型钙离子通道占主导，它参与神经元后去极化电位及膜电位振荡，影响神经元兴奋性。因此，Cav3.2 T型钙离子通道参与神经病理痛的机制受到重视。但是在不同神经病理痛模型，Cav3.2 T型钙离子通道电流和含量及行为药理学结果等相互矛盾。分析文献并结合预实验结果，我们发现，出现上述矛盾的原因可能在于：已有研究没有区分损伤与未损伤的背根神经节（DRG）神经元和外周纤维。目的：采用坐骨神经分支选择性损伤（SNI）模型，以损伤或未损伤的DRG神经元及外周纤维为研究对象，阐明Cav3.2 T型钙离子通道参与神经病理痛的分子机制。方法：采用全细胞膜片钳技术、神经单纤维放电记录技术、分子生物学并结合行为药理学等方法。结果：（1）SNI大鼠模型行为学变化：选择性损伤腓总神经和胫神经，保留腓肠神经的完整，建立SNI模型。SNI术后1 d至28 d，大鼠出现明显的机械痛敏现象。（2）SNI大鼠DRG神经元兴奋性变化：在小直径和直径较小的中等直径神经元，未损伤神经元的兴奋性明显增加，而损伤神经元的兴奋性无明显改变。（3）SNI大鼠损伤的DRG神经元出现Cav3.2通道上膜现象。（4）SNI大鼠损伤和未损伤的外周神经，Cav3.2通道表达上调。（5） 与Sham组相比，感受野给予Mibefradil，能够更明显的增加SNI大鼠腓肠神经的A类单纤维机械阈值。（6）鞘内给予Cav3.2反义寡核苷酸，可以降低未损伤神经纤维上Cav3.2通道，并且缓解神经病理痛。（7）腓肠神经旁给予mibefradil缓解神经病理痛。结论：未损伤神经纤维上Cav3.2 T型钙离子通道参与神经病理痛的发病机制。科学意义：本项目也为外周神经旁给予钙离子通道特异性阻断剂治疗神经病理痛提供理论。