Deleterious effects of discography radiocontrast solution on human annulus cell in聽vitro: changes in cell viability, proliferation, and apoptosis in exposed cells

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• 作者：Helen E. Gruber ; PhD^a ; ^{helen.gruber@carolinashealthcare.org} ; Alfred L. Rhyne III ; MD^b ; Kristopher J. Hansen ; BS^a ; Ryan C. Phillips ; BS^a ; Gretchen L. Hoelscher ; MS^a ; Jane A. Ingram ; BS^a ; H. James Norton ; PhD^c ; Edward N. Hanley Jr. ; MD^a
• 关键词：Disc degeneration ; Discography ; Radiocontrast solution ; Cell death ; Apoptosis
• 刊名：The Spine Journal
• 出版年：2012
• 期刊代码：243_15299430
• 类别：med
• 出版时间：April, 2012
• 卷：12
• 期：4
• 页码：329-335
• 文件大小：707 K

摘要

Background context

Carragee et聽al. have recently shown that modern discography injections are associated with subsequent acceleration of disc degeneration, herniation, and loss of disc height. Although needle puncture and pressurization are known trauma events that can create disc degeneration in animal models, another likely culprit in clinical discography-associated degeneration is a direct effect of the contrast agent itself on disc cells.

Purpose

To test the hypothesis that discography contrast solution would have a deleterious effect on human annulus cells in聽vitro.

Study design

An in聽vitro study using cultured human annulus cells to assay cell death, cell proliferation, and apoptosis.

Patient sample

Annulus cells from eight surgical disc specimens were evaluated (two Thompson Grade III discs and six Grade IV discs) for cell death and proliferation, and an additional five cultures were tested for apoptosis.

Outcome measures

The proportion of dead and live cells, cell proliferation, and the proportion of apoptotic cells in control and experimental groups.

Methods

After internal review board approval, experimental design used two sets of controls: untreated cells under our normal culture conditions (control) and a set with added glucose to adjust the osmolality to match respective Isovue radiocontrast solution treatments (glucose controls) using a freezing point osmometer. Treated cells received Isovue 200 (iopamidol, Isovue-M 200; Bracco Diagnostics, Inc., Princeton, NJ, USA) at 12.5, 25, 50, or 100 mg/mL. Twenty thousand cells/well were seeded in triplicate in 24 well plates, control or test media added, and incubated for 24 hours. At termination, dead cells were identified with trypan blue staining and percentage dead cells determined. Cells were also tested to determine the percentage of apoptotic cells after 50 or 100 mg/mL Isovue exposures. Proliferation assays used standard plate reader methods. Statistical analysis used repeated measures analysis of variance with SAS software (version 9.2; SAS Institute, Inc., Cary, NC, USA).

Results

Analysis of cell death showed a significant increase in the percentage of dead cells with increasing Isovue concentrations compared with control cells (p=.018-.0008). Cell proliferation analyses showed significantly reduced division in Isovue-treated cells (p=.004), and apoptosis assays revealed a significantly higher proportion of apoptotic cells in cells exposed to 50 and 100 mg/mL Isovue (p=.016 and .0003, respectively).

Conclusions

Discography is used extensively in the evaluation of low back pain. Because the lifetime prevalence of disc degeneration and low back pain is high (80%in the general population), many patients may undergo this procedure. Data presented here show that cells exposed in聽vitro to a radiocontrast agent with adjustments for osmolality have significantly reduced proliferation, increased cell death, and increased programmed cell death (apoptosis). In light of the well-recognized age- and degeneration-related decrease in disc cell numbers, it is possible that radiocontrast exposure may be contributing significantly to disc cell loss with subsequent progression of disc degeneration. Findings presented here provide a plausible cell-based explanation for the previously reported disc degeneration in patients receiving discography contrast solutions.