Ma W, Zheng W H, Kar S, Quirion R
Douglas Hospital Research Center, McGill University, 6875 Boul. LaSalle, Verdun, Quebec, H4H 1R3, Montreal, Canada.
Neuroscience. 2000;99(3):529-39. doi: 10.1016/s0306-4522(00)00226-8.
The mechanism of spinal tolerance to the analgesic effects of opiates is unclear at present. We have reported previously that calcitonin gene-related peptide-like immunoreactivity was significantly increased in primary afferents of the spinal dorsal horn during the development of morphine tolerance, suggesting that changes in the level of pain-related neuropeptides in dorsal root ganglion neurons may be involved [Menard D. P. et al. (1996) J. Neurosci. 16, 2342-2351]. In this study, we investigated if in vitro treatment with morphine can mimic the in vivo findings and induce increases in calcitonin gene-related peptide-like immunostaining in cultured dorsal root ganglion neurons from young (three-month-old) and middle-aged (10-month-old) adult rats. Following a repetitive exposure to morphine sulfate (1, 5, 10 microM) for six days, the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons in cultured dorsal root ganglia from three- and 10-month-old rats was significantly increased. A lower concentration (0.5 microM) of morphine induced these increases only in dorsal root ganglion neurons from middle-aged rats. Morphine treatment was also found to increase the number of calcitonin gene-related peptide-immunoreactive neurons possessing multiple, long branches (i.e. with at least one branch >0.5mm). This apparent increase in the number of calcitonin gene-related peptide- and substance P-immunoreactive neurons observed following morphine treatment was blocked by naloxone, an opiate antagonist, indicating the involvement of genuine opioid receptors. No significant change in the number of neuropeptide Y- or galanin-immunoreactive neurons in cultured dorsal root ganglia was detected following any of these treatments. These data suggest that repeated exposure to morphine rather selectively increases calcitonin gene-related peptide- and substance P-like immunoreactivity in cultured dorsal root ganglion neurons. Moreover, the sensitivity to morphine-induced changes is greater in cultured dorsal root ganglion neurons from 10- compared to three-month-old rats. Hence, cultured dorsal root ganglion neurons can provide a model to investigate the cellular and molecular mechanisms underlying alterations in neuropeptide levels following repeated exposure to opiates and their relevance to the development of opioid tolerance.
目前，脊髓对阿片类药物镇痛作用产生耐受的机制尚不清楚。我们之前曾报道，在吗啡耐受形成过程中，脊髓背角初级传入纤维中降钙素基因相关肽样免疫反应性显著增加，这表明背根神经节神经元中与疼痛相关的神经肽水平变化可能与之有关[梅纳德·D·P等人（1996年）《神经科学杂志》16卷，2342 - 2351页]。在本研究中，我们探究了体外给予吗啡是否能模拟体内研究结果，并诱导幼年（三个月大）和中年（十个月大）成年大鼠培养的背根神经节神经元中降钙素基因相关肽样免疫染色增加。在重复暴露于硫酸吗啡（1、5、10微摩尔）六天后，来自三个月和十个月大鼠的培养背根神经节中降钙素基因相关肽和P物质免疫反应性神经元的数量显著增加。较低浓度（0.5微摩尔）的吗啡仅在中年大鼠的背根神经节神经元中诱导了这些增加。还发现吗啡处理增加了具有多个长分支（即至少有一个分支>0.5毫米）的降钙素基因相关肽免疫反应性神经元的数量。吗啡处理后观察到的降钙素基因相关肽和P物质免疫反应性神经元数量的这种明显增加被阿片拮抗剂纳洛酮阻断，表明真正的阿片受体参与其中。在这些处理后的任何一种情况下，培养背根神经节中神经肽Y或甘丙肽免疫反应性神经元的数量均未检测到显著变化。这些数据表明，重复暴露于吗啡能相当选择性地增加培养背根神经节神经元中降钙素基因相关肽和P物质样免疫反应性。此外，与三个月大的大鼠相比，十个月大的大鼠培养背根神经节神经元对吗啡诱导变化的敏感性更高。因此，培养的背根神经节神经元可以提供一个模型，用于研究重复暴露于阿片类药物后神经肽水平改变的细胞和分子机制及其与阿片耐受形成的相关性。
