Hirahara Mikiko, Fujiwara Naoshi, Seo Kenji
Division of Dental Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata City 951-8514, Japan.
Division of Medical Technology, Niigata University Graduate School of Health Sciences, Japan.
J Neurosci Methods. 2017 Jul 15;286:6-15. doi: 10.1016/j.jneumeth.2017.05.019. Epub 2017 May 15.
The trigeminal subnucleus caudalis (Vc) plays a critical role in transmission and modulation of nociceptive afferent inputs, and exhibits a similar layer construction to the spinal dorsal horn. However, afferent inputs enter the brainstem and project to a separately located nucleus. It has previously been difficult to record responses of the Vc to afferent fiber activation in a brainstem slice preparation. The aim of the present study was to establish a novel brainstem slice preparation method to study trigeminal nociceptive transmission mechanisms.
Thirty adult 6-7-week-old C57/BL6J male mice were included in the study. Obliquely sliced brainstem sections at a thickness of 600μm, which included the Vc and the root entry zone to the brainstem, were prepared. The Vc response to electrical stimulation of afferent fibers was observed as a change in intracellular calcium concentration by fluorescence intensity response.
Electrical stimulation of afferent inputs to the trigeminal nerve increased fluorescent intensity in the Vc, which was completely diminished by tetrodotoxin and significantly suppressed by the AMPA/kainate antagonist CNQX (paired t-test, P<0.001), although the non-competitive NMDA antagonist (+)-MK801 maleate resulted in no changes. These results suggested a glutamate receptor-mediated response.
COMPARISON WITH EXISTING METHODS/CONCLUSION: This brainstem slice preparation will be useful for investigating nociceptive transmission mechanisms of the trigeminal nerve.
三叉神经尾侧亚核（Vc）在伤害性传入输入的传递和调制中起关键作用，并且表现出与脊髓背角相似的分层结构。然而，传入输入进入脑干并投射到一个单独定位的核团。以前，在脑干切片制备中记录Vc对传入纤维激活的反应一直很困难。本研究的目的是建立一种新的脑干切片制备方法来研究三叉神经伤害性传递机制。
本研究纳入了30只6-7周龄的成年C57/BL6J雄性小鼠。制备了厚度为600μm的斜切脑干切片，其中包括Vc和脑干的神经根进入区。通过荧光强度反应观察Vc对传入纤维电刺激的反应，以细胞内钙浓度的变化来表示。
对三叉神经传入输入的电刺激增加了Vc中的荧光强度，河豚毒素可使其完全消失，AMPA/海人酸拮抗剂CNQX可显著抑制（配对t检验，P<0.001），尽管非竞争性NMDA拮抗剂马来酸（+）-MK801没有引起变化。这些结果提示了一种谷氨酸受体介导的反应。
与现有方法的比较/结论：这种脑干切片制备方法将有助于研究三叉神经的伤害性传递机制。
