Cytotoxic Effect of Commercially Available
Methylprednisolone Acetate with and without
Reduced Preservatives on Dorsal Root Ganglion
Sensory Neurons in Rats
Authors: Knezevic Nebojsa N. 
Published: February 2021

Background: 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.
Objective: 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.
Methods: 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.
Results: 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.
Limitations: 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.
Conclusions: 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 (salinetreated) 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.
Key words: Methylprednisolone acetate, preservatives, dorsal root ganglion sensory neurons,
cytotoxic effect, polyethylene glycol, myristylgamma-picolinium chloride