椎管內注射百日咳毒素動物模式探討神經病理性疼痛之細胞學機轉

Implications of Intrathecal Pertussis Toxin Animal Model on the Cellular Mechanisms of Neuropathic Fain Syndrome

如同類鴉片耐藥性，神經病理性疼痛(neuropathic pain)通常對於類鴉片藥物的鎮痛效果反應並不好，且伴隨著痛覺過敏(hyperalgesia)及觸感痛(allodynia)。有關神經病理性疼痛的發生及預防的機制到目前為止仍不清楚。在我們過去的研究發現椎管內給予百日咳素不僅可以造成嗎啡止痛作用的降低，同時也會誘發對溫度痛覺過敏(thermal hyperalgesia)之行為。百日咳毒素經由對抑制型Gi或Go之鳥嘌呤蛋白之ADP-ribosylation，造成抑制性訊息的傳遞受到抑制，進而使原本抑制性及興奮性兩系統間之平衡受到破壞，結果造成興奮性的訊息傳遞相對地提高。目前有研究報告認為，椎管內注射百日咳毒素可以用來當作一個研究中樞性的神經病理性疼痛之動物模式。在系列的研究中，我們利用椎管內微透析及給藥之技術，探討在大白鼠椎管內注射百日咳毒素後，以脊髓為研究範圍來探討神經病理性疼痛之細胞學機轉。結果發現，在 大白鼠椎管內注射百日咳毒素可以誘發溫度疼痛過敏，且伴隨著脊髓液中興奮性胺基酸麩胺酸(glutamate)及天冬胺酸(aspartate)釋放量之增加，然而抑制性胺基酸甘胺酸(glycine)則明顯下降。但百日咳毒素對其它胺基酸如絲胺酸(serine)、麩醯胺(asparagine)、牛磺酸(taurine)、精胺酸(arginine)及一氧化氮的釋量並沒有影響，由此結果判斷可以去除百日咳毒素對細胞之毒殺作 用。而由百日咳毒素所誘發的疼痛過敏行為可以被NMDA接受器之拮抗劑D-AP5、甘胺酸及蛋白質激酶C(protein kinase C)抑制劑chelerythrine所抑制。同時我們也發現，椎管內注射百曰咳毒素可以誘發脊髓中疼痛相關神經元的細胞質及細胞膜之蛋白質激酶C-γ含量增加，但對α、βI及βII等其它異構酶(isoenzyme)卻沒有影響。而百日咳毒素所造成蛋白質激酶C-γ表現量之增加則受到椎管內D-AP5灌注所抑制，然而D-AP5對正常大白鼠脊髓中蛋白質激酶C-α、βI、βII及γ異構酶則並無影響。由以上系列研究結果顯示，椎管內注射百日咳毒素除了會誘發對溫度痛覺過敏之行為外，並伴隨有脊髓中興奮性胺基酸釋放量增加、抑制性胺基酸減少及蛋白質激酶C表現量之增加。而百日咳毒素所造成之溫度痛覺過敏及嗎啡止痛作用降低之現象，可以受到椎管內給予蛋白質激酶C抑制劑所阻斷。這些實驗結果顯示百日咳毒素所誘發的神經病理性疼痛（溫度痛覺過敏），包含蛋白質激酶C、興奮性及抑制性胺基酸是參與其中之機轉。

Like opioid tolerance, neuropathic pain syndrome manifested by hyperalgesia and allodynia responds poorly to opioids. Hitherto, its development is still not clear and its treatment and prevention are still disputable. Pertussis toxin (PTX) which ADP-ribosylates the α-subunit of inhibitory guanine nucleotide binding regulatory proteins (Gi/Go), is used to induce morphine tolerance through intrathecal (i.t.) injection. It decreases the antinociceptive effect of opioid receptor agonists, and produces a thermal hyperalgesia as well. With treatment of PTX the inhibitory Gi-and Go-proteins signal transduction is inactivated. Inhibition of the inhibitory system would likely lead to a predominance of the excitatory system. Intrathecal PTX administration has also been suggested as a model for study of the central mechanisms of neuropathic pain. In our previous studies, with intrathecal microdialysis and drug delivery techniques, we correlated the biochemical and pharmacological effects on the behavioral expressions of i.t. PTX-treated rats. Intrathecal PTX administration would induce thermal hyperalgesia in rats, with accompaniments of a prolonged increase in the concentrations of excitatory amino acids (EAAs), glutamate and aspartate, and a decrease in the concentration of the inhibitory amino acid (IAA) glycine in the spinal CSF dialysates. The PTX-induced thermal hyperalgesia peaked between day 2 and 4, but no cold allodynia is observed; i.t. administration of N-methyl-D-aspartate (NMDA) receptor antagonist, D-2-amino-5-phosponovaleric acid (D-AP5), glycine and protein kinase C (PKC) inhibitor chelerythrine attenuated the thermal hyperalgesia. The PKCy content of both synaptosomal and cytosolic fractions were significantly increased in PTX-treated rats. In contrast, the levels of PKCα, βI, or βII isozymes in these fractions were unaffected. Infusion of NMDA antagonist D-AP5 prevented both the thermal hyperalgesia and the increase in PKCy expression in PTX-treated rats. Similar to our previous report, i.t. PTX reduced morphine's analgesic effect. PKC inhibitor chelerythrine attenuated this reduction of morphine's analgesia, and an inhibition of the morphine-evoked EAAs release was observed in PTX-treated rats as well. Taken together, i.t. PTX-induced neuropathic pain syndrome is accompanied by increasing of EAAs, decreasing of IAA release, and a selective increasing of PKCy expression in the spinal cord. Inhibition of PKC not only blocked thermal hyperalgesia, but also reversed the reduction of morphine's analgesic effect in PTX-rats. These results suggest that PTX-induced neuropathic pain syndromes are involved in EAAs, IAAs and PKC alternations.