Eaton M J, Plunkett J A, Karmally S, Martinez M A, Montanez K
The Miami Project To Cure Paralysis, University of Miami School of Medicine, FL 33136, USA.
J Chem Neuroanat. 1998 Dec;16(1):57-72. doi: 10.1016/s0891-0618(98)00062-3.
We have utilized RN46A cells, an immortalized neuronal cell line derived from E13 brainstem raphe, as a model for transplant of bioengineered serotonergic cells. RN46A cells require brain-derived neurotrophic factor (BDNF) for increased survival and serotonin (5HT) synthesis in vitro and in vivo. RN46A cells were transfected with the rat BDNF gene, and the 46A-B14 cell line was subcloned. These cells survive longer than 7 weeks after transplantation into the subarachnoid space of the lumbar spinal cord and synthesize 5HT and BDNF. Chronic constriction injury (CCI) of the sciatic nerve was used to induce chronic neuropathic pain in the affected hindpaw in rats. Transplants of 46A-B14 cells placed 1 week after CCI alleviated chronic neuropathic pain, while transplants of 46A-V1 control cells, negative for 5HT and without the BDNF gene, had no effect on the induction of thermal and tactile nociception. When endogenous cells of the dorsal horn which contain the neurotransmitter gamma-aminobutyric acid (GABA) and its synthetic enzyme glutamate decarboxylase (GAD) were immunohistochemically quantified in the lumbar spinal cord 3 days and 1-8 weeks after CCI, the number of GABA- and GAD-immunoreactive (ir) cells decreased bilateral to the nerve injury as soon as 3 days after CCI. At 1 week after CCI, the number of GABA-ir cells continued to significantly decline bilaterally, returning to near normal numbers on the side contralateral to the nerve injury by 8 weeks after the nerve injury. The number of GAD-ir cells began to increase bilaterally to the nerve injury at 1 week after CCI and continued to significantly increase in numbers over normal values by 8 weeks after the nerve injury. When examined 2 and 8 weeks after CCI plus cell transplants, the transplants of 46A-B14 cells reversed the increase in GAD-ir cell numbers and the decrease in GABA-ir cells by 1 week after transplantation, while 46A-V1 control cell transplants after CCI had no effect on the changes in numbers of GAD-ir or GABA-ir cells. Collectively, these data suggest that altered 5HT levels, and perhaps BDNF secretion, related to the transplants ameliorate chronic pain and reverse the induction and maintenance of an endogenous pain mechanism in the dorsal horn. This induction mechanism is likely dependent on altered GAD regulation and GABA synthesis, initiated by CCI.
我们利用RN46A细胞，一种源自E13脑干中缝的永生化神经元细胞系，作为生物工程化血清素能细胞移植的模型。RN46A细胞在体外和体内都需要脑源性神经营养因子（BDNF）来提高存活率和血清素（5HT）合成。用大鼠BDNF基因转染RN46A细胞，并亚克隆出46A - B14细胞系。将这些细胞移植到腰段脊髓蛛网膜下腔后存活超过7周，并合成5HT和BDNF。坐骨神经慢性压迫损伤（CCI）用于诱导大鼠患侧后爪的慢性神经性疼痛。CCI后1周进行的46A - B14细胞移植减轻了慢性神经性疼痛，而5HT阴性且无BDNF基因的46A - V1对照细胞移植对热痛觉和触觉痛觉的诱导没有影响。当在CCI后3天以及1 - 8周对腰段脊髓中含有神经递质γ-氨基丁酸（GABA）及其合成酶谷氨酸脱羧酶（GAD）的背角内源性细胞进行免疫组织化学定量分析时，CCI后3天，神经损伤双侧的GABA和GAD免疫反应性（ir）细胞数量就开始减少。在CCI后1周，双侧GABA - ir细胞数量继续显著下降，在神经损伤后8周，神经损伤对侧的细胞数量恢复到接近正常水平。CCI后1周，GAD - ir细胞数量开始在神经损伤双侧增加，并在神经损伤后8周持续显著高于正常数量。在CCI加细胞移植后2周和8周进行检查时，46A - B14细胞移植在移植后1周逆转了GAD - ir细胞数量的增加和GABA - ir细胞数量的减少，而CCI后的46A - V1对照细胞移植对GAD - ir或GABA - ir细胞数量的变化没有影响。总体而言，这些数据表明，与移植相关的5HT水平改变以及可能的BDNF分泌改善了慢性疼痛，并逆转了背角内源性疼痛机制的诱导和维持。这种诱导机制可能依赖于CCI引发的GAD调节和GABA合成改变。
