Department of Anesthesiology, Advocate Illinois Masonic Medical Center, Chicago, IL; Department of Anesthesiology, University of Illinois, Chicago, IL; Biomedical Imaging Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA; Department of Phar.
Pain Physician. 2014 Sep-Oct;17(5):E609-18.
Epidural and intrathecal injections of methylprednisolone acetate (MPA) have become the most commonly performed interventional procedures in the United States and worldwide in the last 2 decades. However neuraxial MPA injection has been dogged by controversy regarding the presence of different additives used in commercially prepared glucocorticoids. We previously showed that MPA could be rendered 85% free of polyethylene glycol (PEG) by a simple physical separation of elements in the suspension.
The objective of the present study was to explore a possible cytotoxic effect of commercially available MPA (with intact or reduced preservatives) on rat sensory neurons.
We exposed primary dissociated rat dorsal root ganglia (DRG) sensory neurons to commercially available MPA for 24 hours with either the standard (commercial) concentration of preservatives or to different fractions following separation (MPA suspension whose preservative concentration had been reduced, or fractions containing higher concentrations of preservatives). Cells were stained with the TUNEL assay kit to detect apoptotic cells and images were taken on the Bio-Rad Laser Sharp-2000 system. We also detected expression of caspase-3, as an indicator of apoptosis in cell lysates.
We exposed sensory neurons from rat DRG to different concentrations of MPA from the original commercially prepared vial. TUNEL assay showed dose-related responses and increased percentages of apoptotic cells with increasing concentrations of MPA. Increased concentrations of MPA caused 1.5 - 2 times higher caspase-3 expression in DRG sensory neurons than in control cells (ANOVA, P = 0.001). Our results showed that MPA with reduced preservatives caused significantly less apoptosis observed with TUNEL assay labeling (P < 0.001) and caspase-3 immunoblotting (P = 0.001) than in neurons exposed to MPA from a commercially prepared vial or "clear phase" that contained higher concentrations of preservatives. Even though MPA with reduced preservatives caused 12.5% more apoptosis in DRG sensory neurons than in control cells, post hoc analysis showed no differences between these 2 groups.
Our data was collected from in vitro isolated rat DRG neurons. There is a possibility that in vivo neurons have different extents of vulnerability compared to isolated neurons.
Results of the present study identified a cytotoxic effect of commercially available MPA with preservatives or with a "clear phase" containing higher concentrations of preservatives on primary isolated rat DRG sensory neurons. This was shown by TUNEL positive assay and by increased caspase-3 expression as one of the final executing steps in apoptotic pathways in DRG neurons. However, our results showed no statistically significant difference between the control cells (saline-treated) and cells treated with MPA with reduced concentrations of preservatives, pointing out that either PEG or myristylgamma-picolinium chloride (MGPC) or their combination have harmful effects on these cells. Reduction of concentrations of preservatives from commercially available MPA suspensions by using the simple method of inverting vials for 2 hours could be considered useful in clinical practice to enhance the safety of this depot steroid when injected neuraxially.
在过去的 20 年中，硬膜外和鞘内注射醋酸甲泼尼龙（MPA）已成为美国和全球最常进行的介入性手术。然而，由于商业上制备的糖皮质激素中存在不同的添加剂，神经轴突 MPA 注射一直存在争议。我们之前的研究表明，通过简单的物理分离悬浮液中的元素，MPA 可以 85%地去除聚乙二醇（PEG）。
本研究的目的是研究市售 MPA（具有完整或减少的防腐剂）对大鼠感觉神经元的可能细胞毒性作用。
我们将原代分离的大鼠背根神经节（DRG）感觉神经元暴露于市售 MPA 24 小时，其防腐剂浓度为标准（商业）浓度，或分离后的不同浓度（防腐剂浓度降低的 MPA 悬浮液，或含有更高浓度防腐剂的）。用 TUNEL 检测试剂盒对细胞进行染色，以检测凋亡细胞，并在 Bio-Rad LaserSharp-2000 系统上拍摄图像。我们还检测了细胞裂解物中 caspase-3 的表达，作为细胞凋亡的指标。
我们将大鼠 DRG 的感觉神经元暴露于原始市售小瓶中的不同浓度的 MPA。TUNEL 检测显示，随着 MPA 浓度的增加，细胞凋亡的比例呈剂量相关性增加。与对照组细胞相比，高浓度的 MPA 导致 DRG 感觉神经元中 caspase-3 的表达增加了 1.5-2 倍（ANOVA，P=0.001）。我们的结果表明，与用市售小瓶或含有较高浓度防腐剂的“澄清相”制备的 MPA 相比，用减少防腐剂的 MPA 处理的神经元，用 TUNEL 检测标记观察到的细胞凋亡明显减少（P<0.001），用 caspase-3 免疫印迹检测也明显减少（P=0.001）。尽管用减少防腐剂的 MPA 处理的 DRG 感觉神经元比对照组细胞的凋亡率高 12.5%，但事后分析显示这两组之间没有差异。
我们的数据来自体外分离的大鼠 DRG 神经元。与分离神经元相比，体内神经元可能具有不同程度的易感性。
本研究结果表明，市售 MPA 及其防腐剂或含有较高浓度防腐剂的“澄清相”对原代分离的大鼠 DRG 感觉神经元具有细胞毒性作用。这一点通过 TUNEL 阳性检测和 caspase-3 表达的增加得到证实，caspase-3 是 DRG 神经元凋亡途径中的最后一个执行步骤之一。然而，与用生理盐水处理的对照组细胞相比，用减少防腐剂浓度的 MPA 处理的细胞之间没有统计学上的显著差异，这表明 PEG 或肉豆蔻基γ-皮考啉氯盐（MGPC）或它们的组合对这些细胞有有害影响。通过简单地颠倒小瓶 2 小时来减少市售 MPA 悬浮液中的防腐剂浓度，可以考虑在临床实践中使用，以提高神经轴突内注射这种储存类固醇的安全性。
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