Superparamagnetic Iron Oxide–Enhanced Magnetic Resonance Imaging of Neuroinflammation in a Rat Model of Radicular Pain

In many clinical cases of radicular pain, no noticeable neuropathology is detected by conventional medical imaging strategies. Superparamagnetic iron oxide (SPIO) nanoparticles were evaluated as magnetic resonance contrast agents to specifically detect neuroinflammation at sites of painful injury in a rat model of cervical nerve root compression. Two separate groups of rats were used: an injury group that underwent controlled transient compression of the dorsal root and a sham group that received the same surgical procedures but no injury. Precontrast magnetic resonance imaging (MRI) was performed 6 days after surgery, followed by administration of SPIO via tail vein injection. After 24 hours, T 2 * -weighted imaging at the site of root injury revealed a postcontrast enhancement of 72.9 ± 31%. This was significantly greater than that of injured animals prior to SPIO administration (5.3 ± 12.9%). SPIO did not generate any significant postcontrast enhancement in the nerve roots of the sham group. Histology confirmed colocalization of SPIO with macrophage at the injury site. These findings suggest that SPIO-enhanced MRI may be a valuable tool to identify otherwise undetectable nerve root compression and enable improved patient management

Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1β and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These results are the first to demonstrate that salmon thrombin has unique analgesic, neuroprotective and anti-inflammatory capabilities compared to human thrombin and that PAR1 may contribute to these actions

Myelin basic protein (MBP) organization in the injured nerve root.

<p>−striated MBP patterning;</p><p>+mostly striated MBP with some disruption in labeling;</p><p>++mostly disorganized MBP labeling;</p><p>+++highly disorganized MBP labeling with pockets of debris.</p

Salmon and human thrombin have similar affinities for serum containing media and ATIII, but not hirudin.

<p>(<b>A</b>) Salmon thrombin (STh) and human thrombin (HTh) maintain similar enzymatic activity towards a fluorescent fibrinogen-like substrate over time in serum containing media kept at 37°C; their normalized cleavage rate is not different at any time point after thrombin addition. (<b>B</b>) The activity of salmon thrombin is inhibited significantly less (p = 0.005) than human thrombin by Antithrombin III (ATIII) overall, but not at any one individual concentration ranging from 0 to 45 nM. (<b>C</b>) Salmon thrombin activity towards fibriniogen is inhibited less than human thrombin overall (p<0.001) and at hirudin-to-thrombin ratios of 1 and 1.5 (*p<0.001). Data are shown as means with standard deviations (μ ± SD).</p

Salmon thrombin does not increase pro-inflammatory cytokines in cortical cultures at early time points.

<p>(<b>A</b>) IL-1β and TNFα mRNA are significantly upregulated in cortical cultures at 4 hours after their treatment with human thrombin (HTh) at 1 U/ml compared to untreated cultures (UT) (^#p<0.001). Salmon thrombin (STh), at the same concentration, is unchanged from UT and significantly less than human thrombin treatment (HTh) (*p<0.002). (<b>B</b>) IL-6 concentration is significantly greater in cultures treated with human thrombin overall at each individual concentration (^#p<0.01) for 6 hours compared to the untreated (UT) control. IL-6 secreted from cortical cultures is significantly less after treatment with salmon thrombin (STh) compared to human thrombin (HTh) at 6 hours after stimulation overall (p<0.001) and at each individual concentration of 0.2, 0.5 and 1 U/ml (*p<0.03). Data are shown as means with standard deviations (μ ± SD).</p

Salmon thrombin preserves nerve root health and prevents inflammation after painful compression in the rat.

<p>(<b>A</b>) Schematic depicting the spinal cord, nerve root and dorsal root ganglion (DRG); red box indicates location within the nerve root where the root was analyzed. (<b>B</b>) Uncompressed nerve roots from un-operated (normal) rats exhibit myelin basic protein (MBP; green) labeling in a striated pattern that is homogenous across the width of the root and no immunoreactivity for macrophages (Iba1; red). On day 7 after a painful root compression treated with neurobasal media (NB media), MBP is disrupted and Iba1 is more abundant. Human thrombin treated roots (HTh) have MBP and Iba1 labeling that is similar to those roots treated with NB media. Salmon thrombin treated roots (STh) exhibit the same striated MBP labeling with minimal Iba1 as normal roots and much less than roots treated with NB media or human thrombin. Scale bar is 100 µm. (<b>C</b>) Quantification of positive Iba1 labeling normalized to expression in normal un-operated tissue. Normal tissue exhibits very low levels of Iba1. Human thrombin significantly increases (^#p<0.001) Iba1 in the nerve root compared to normal. Roots treated with salmon thrombin are not different from normal levels or those treated with neurobasal media, but induces significantly less (*p = 0.035) Iba1 infiltration in the nerve root compared to human thrombin.</p