正向調節三叉神經節之 alpha6-GABAA受體減緩硝化甘油重覆注射所誘發之小鼠偏頭痛表情

Abstract

偏頭痛是一種高盛行率的失能疾病。這種神經系統疾病背後的機制尚未被完全闡明，加上目前的藥物並未能滿足治療上的需求或是具有無法忍受的副作用，這使得偏頭痛成為難以治療的疾病之一。因此，探究偏頭痛的病生理學，並開發對偏頭痛治療幾乎沒有副作用的臨床有效藥物是有其迫切之需要。建立能夠模擬偏頭痛臨床表現的動物行為模式將有助於了解其機制並開發新的治療方法。 在本研究中，我們建立了一個小鼠的偏頭痛行為模式。此模式透過重複給予小鼠全身性注射會產生頭痛副作用的硝酸甘油（Nitroglycerin, NTG）來誘發頭痛症狀，並以偏頭痛般的疼痛指標，包括皺眉，鼻子噘起，臉頰鼓起，以及耳朵位置和鬍鬚的變化，分別以0(無疼痛反應)到2(明顯疼痛反應)的小鼠疼痛評估量表作為疼痛評分標準來探究NTG造成小鼠頭痛的嚴重程度。 NTG（10 mg / kg，ip）會在9天中每兩天注射一次（共5個試驗）。結果顯示重複在小鼠身上給予NTG能誘發顯著的似偏頭痛的疼痛反應，此疼痛反應都能穩定的在每次NTG注射後的30至60分鐘顯現。值得一提的是，NTG所誘發出的疼痛表情能夠透過臨床上用來治療偏頭痛急性期症狀的舒馬曲坦(sumatriptan)以及預防性抗偏頭痛藥物托吡酯(Topiramate)消除和預防。 在這個重複注射NTG誘導偏頭痛模式中，我們測試了對含有α6亞型γ-氨基丁酸A受體（α6GABAAR）具選擇性的兩種正向異位調節劑（PAM）的作用。在急性頭痛症狀療效評估方面，於第5次注射NTG後20分鐘給予Compound 6（3和10 mg / kg，ip）及其等效和長效氘衍生物DK-I-56-1（3和10 mg / kg，ip）均顯著舒緩了此偏頭痛模型小鼠因NTG所致的疼痛表情。此外，Compound 6和DK-I-56-1的作用能夠為呋塞米（20mg / kg，i.p），一種α6GABAAR-選擇性拮抗劑所拮抗。同時，我們也探究α6GABAAR PAMs在預防偏頭痛中的作用。在五次NTG注射前30分鐘給予Compound 6，DK-I-56-1和臨床預防偏頭痛發作藥物托吡酯來評估其偏頭痛預防之成效。有趣的是，試驗中採用的兩種α6GABAAR PAMs中，僅DK-I-56-1 ( 3mg / kg，i.p）展現有效預防NTG所誘發的偏頭痛樣疼痛反應，且其效力可與30mg / kg托吡酯（i.p）比擬。另外，我們還發現DK-I-56-1（10 mg / kg）通過口服仍保留其抗偏頭痛特性。免疫螢光染色顯示α6GABAARs大量分佈於小鼠三叉神經元和衛星膠質細胞中。總之，這些結果表明α6GABAAR PAM對於減緩偏頭痛疼痛表徵或預防性治療是有效的，並且其作用標的坐落在周邊神經(例如:三叉神經節)上。 此外，我們取出經NTG重複注射的小鼠之三叉神經節(Trigeminal ganglia, TG)進行切片並執行免疫螢光染色以研究在TG中的γ-氨基丁酸(GABA)系統平衡是否受到影響。結果顯示，重複的NTG處理並不影響神經細胞中α6GABAARs和GABA轉運蛋白1（GAT1）的表達水平。然而，在重複暴露於NTG後，小鼠的TG神經細胞中65kDa谷氨酸脫羧酶（GAD65）(三叉神經節中的GABA合成酶）的表達水平顯著降低。綜合上述觀察，重複的NTG給藥可能會透過使GAD 65的表現量減低而讓GABA-α6GABAAR神經傳遞受損，進而降低三叉神經節中的GABA水平，最終產生偏頭痛。由於偏頭痛並沒有改變三叉神經節中 α6GABAAR的含量，因此它們可以作為偏頭痛的治療靶點。就以Compound 6和DK-I-56-1而言，對三叉神經節中的α6GABAAR的選擇性正向調節可以恢復受損的TG GABA系統的傳遞，帶來偏頭痛緩解。這些發現可能為偏頭痛的治療開闢了一條新途徑。

Migraine is a debilitating disorder with high prevalence. The mechanism behind this neurological disorder is still not yet fully elucidated and current medications are either not fulfill the treatment requirement or have intolerable side effects, which make migraine to be one of unmet medical needs. Therefore, exploring the pathophysiology of migraine to develop clinically effective agents with little side effects for migraine is an urgent need. To establish an animal behavioral model that can mimic clinical manifestations of migraine would be benefit to understanding its mechanism and developing new treatments. In the present study, we have established a mouse model of migraine by giving mice repetitive systemic injections with nitroglycerin (NTG), a vasodilator known for its headache side effect, and assessing the severity of migraine in mice by their grimaces (facial expression) NTG (10 mg/kg, i.p.) was injected into mice once every two days in 9 days (5 sessions). The migraine-like grimaces, including orbital tightening, nose bulge, cheek bulge, and the changes of ear position and whisker were scored from 0-2 as the mouse grimace score. Repeated NTG administrations in mice induced significant migraine-like grimaces, which were consistently emerged in between 30 to 60 minutes after NTG administrations. Importantly, NTG-induced grimaces were abolished and prevented by sumatriptan and topiramate, clinically used abortive and preventive anti-migraine medications, respectively. In this repeated NTG administrations-induced migraine model, we tested effects of two positive allosteric modulators (PAMs) selectively targeting at the α6 subunit-containing GABAA receptors (α6GABAARs). As abortive treatments, drugs were administered 20 min after the 5th NTG injection. Both Compound 6 (3 and 10 mg/kg, i.p.), and its equipotent and longer-acting deuterated derivative, DK-I-56-1 (3 and 10 mg/kg, i.p.) significantly attenuated the elevated grimace score in this migraine model. Moreover, effects of Compound 6 and DK-I-56-1 were antagonized by furosemide (20 mg/kg, i.p.), an α6GABAA R-selective antagonist. In addition, we also examined effects of α6GABAAR PAMs in migraine prevention. Compound 6, DK-I-56-1 and a reference drug, topiramate, were given 30 min before NTG injection in each of the five NTG-injection sessions. Interestingly, DK-I-56-1, but not Compound 6, at 3 mg/kg (i.p.), was also effective in preventing NTG-induced migraine-like grimace with the efficacy comparable to 30 mg/kg topiramate (i.p.). Additionally, we also found DK-I-56-1 (10 mg/kg) retained its anti-migraine property through oral administration. Immunofluorescence staining showed that α6GABAARs are abundantly distributed in neurons and satellite glial cells in mouse TG. Together, these results suggest that α6GABAAR PAMs are effective as either an abortive or preventive treatment for migraine and also possess peripheral action site. We also performed immunofluorescent staining on TG sections of NTG-repetitively-treated mice to investigate if the GABAergic homeostasis is affected. The results demonstrated that repeated NTG treatments did not affect the expression level of α6GABAARs, nor the GABA transporter 1 (GAT1) in TG neuronal cells and satellite glial cells. However, the expression level of 65 kDa Glutamate decarboxylase (GAD65), a GABA synthesizing enzyme in TG, was striking lower in TG neuronal cells of mice after repeated exposure to NTG. To sum up, repeated NTG administrations may lead to lower GABA levels, due to downregulation of GAD 65, and impaired GABA-α6GABAAR neurotransmission in TG, resulting in migraine. Since migraine did not alter the level of TG α6GABAARs, they may be served as the therapeutic target of migraine. Selective positive modulation of the TG α6GABAARs, in the case by Compound 6 and DK-I-56-1, may restore the impaired TG GABAergic transmission, leading to migraine alleviation. These findings may open up an exciting new avenue for the novel treatment of migraine