神經毒素(resiniferatoxin)造成之週邊神經退化與4-methylcatechol促進表皮神經再生之研究

Abstract

在本實驗中，我們利用具有高專一性破壞小直徑感覺神經的神經毒素(resiniferatoxin, RTX)來引發小直徑感覺神經受到損傷的週邊神經性疼痛模式。單一劑量的RTX水溶液經由腹腔注射後(50μg/kg)七天，可以觀察到表皮上的小直徑感覺神經明顯的減少(p = 0.0067)；相較於控制組(vehicle group)，約略減少了66%。同時經由電子顯微鏡的超微結構的分析顯示，表皮小直徑感覺神經的減少是因為無髓鞘神經退化之故；其無髓鞘神經密度減少約53%。此外，表皮小直徑感覺神經減少也同時造成了該些實驗小鼠對於熱刺激反射時間明顯的增加以及機械性刺激的閾值降低。 免疫染色的結果顯示，具有calcitonin gene-relative peptide (CGRP) 與substance p (SP) 免疫反應的表皮小直徑感覺神經有不同程度的減少，此一現象可以由具有CGRP與SP免疫反應的背根神經結細胞本體也具有不同程度的減少得到一致性的驗證。此外，大直徑運動與感覺神經纖維則不受RTX的影響。 施打resiniferatoxin後第七天，開始每天經由腹腔注射4-methylcatechol (4MC, 10μg/kg)直至第三十五天的實驗小鼠顯示施打4MC的實驗小鼠其無髓鞘神經密度有明顯的增加(p = 0.014)；此一現象則更進一步具體的表現在表皮小直徑感覺神經密度的增加(p = 0.0013)。熱刺激與機械性刺激的數據也顯示施打4MC的小鼠其熱刺激的反應時間明顯縮短以及機械性刺激的閾值較高。 長期施打4MC至注射RTX後第56天及第84天顯示protein gene product 9.5(PGP 9.5)(+)及CGRP(+)的表皮神經密度有顯著且較快速度的再生；惟對於SP(+)的表皮神經則沒有明顯效用。綜合以上結果可以得知，實驗小鼠經由RTX注射後，會造成無髓鞘神經退化以及表皮去神經支配；此一神經病理變化同時會伴隨著熱刺激感覺喪失以及降低機械性刺激的閾值。4MC注射後，可以明顯的促進無髓鞘神經再生以及表皮神經的再支配，並讓熱刺激以及機械性刺激的感覺回復。

To generate an experimental neuropathy specifically affecting small-diameter sensory nerves, we treated mice with a capsaicin analogue, resiniferatoxin (RTX), through a single intraperitoneal injection (50 μg/kg). On day 7 (D7) after RTX treatment compared to the vehicle-treated group, unmyelinated nerves of the medial plantar nerves in the RTX group showed significant degeneration with skin denervation in the corresponding territory as evidenced by a 53% reduction in unmyelinated nerve density of medial plantar nerve (p = 0.0067) and a 66% reduction in epidermal nerve density of hindpaw skin (p = 0.0004). These changes were associated with functional deficits of prolonged withdrawal latencies to heat stimuli (p = 0.0007) on a hot plate test and reduced mechanical threshold (p = 0.0001). Immunoreactive for calcitonin gene-relative peptide (CGRP) and substance P (SP) epidermal nerves were different degree depleted and those confirmed by the mild depletion of dorsal root ganglion neurons immunoreactive for CGRP (p = 0.005) and markedly depleted for SP (p = 0.0001). Large-diameter motor and sensory nerves were not affected as assayed by nerve conduction studies and sural morphometric study showed no affected on the large and small-diameter myelinated sensory nerves. We then investigated the potential therapeutic effect of 4-methylcatechol (4MC) through a daily intraperitoneal injection of 4MC (10 μg/kg) from D7 to D35 after RTX-induced neuropathy. On D35, 4MC significantly promoted regeneration of unmyelinated nerves as demonstrated by an increase in unmyelinated nerve density (p = 0.014) with an increase in the epidermal nerve density (p = 0.0013) and a reduction in the thermal withdrawal latency (p = 0.0091) compared to the RTX group. Long-term of 4MC-treated only accelerated the reinnervation of PGP 9.5 and CGRP-immunreactive epidermal fibers, with SP-immunoreactive fibers remaining dereased. These findings indicate that 4MC promoted regeneration of unmyelinated nerves and accelerated the skin reinnervtion after RTX-induced neuropathy. Moreover, 4MC also reduced the duration of loss thermal responses and the reducing the mechanical thresholds.