Abstract
Summary Statement
In this study, we observed that MR16-1, an interleukin-6 inhibitor, recovered phosphatidylcholine containing docosahexaenoic acid at the injury site after spinal cord injury in mice model by using imaging mass spectrometry.
Introduction
The current drugs for improving motor function of the limbs lost due to spinal cord injury (SCI) are ineffective. Development of new drugs for spinal cord injury is desired. MR16-1, an interleukin-6 inhibitor, is found to be effective in improving motor function after spinal cord injury in mice model. Thus, we examined the molecular mechanism in more detail. Therefore, the purpose of this study was to analyze the molecular changes in the spinal cord of the SCI mice treated with MR16-1 using imaging mass spectrometry.
Methods
All experiments were performed according to the guidelines for animal experimentation and care and use of laboratory animals established by Hamamatsu University School of Medicine (Shizuoka, Japan). We used 36 adult female C57BL/6J mice for laminectomy and contusion injury of the spinal cord that were performed at the T10 level using the Infinite Horizon Impactor (IH Impactor, 60 kdyn; Muromachi, Tokyo, Japan). Immediately after SCI, mice were intraperitoneally injected with a single dose of MR16-1 (Chugai, Tokyo Japan) (100 µg/g body weight, MR16-1 group) or a single dose of phosphate-buffered saline (PBS) of the same volume (control group). Motor function of the hind limbs was evaluated using the Basso Mouse Scale (BMS), an open-field locomotor test in which the scores range from 0 points (scored for no ankle movement) to 9 points (scored for complete functional recovery). BMS scores were recorded at 1, 7, 14, 21, 28, 35, and 42 days after SCI. The spinal cord tissues were flash frozen and were sliced to a thickness of 8 µm using a cryostat (CM1950; Leica, Wetzler, Germany). Imaging mass spectrometry was used to visualise 12 molecular species of phosphatidylcholine (PC) from thin slices of the spinal cords obtained at 7 days post-SCI.
Results
The contusive SCI immediately resulted in complete paralysis. The MR16-1–treated group showed a significant improvement in the BMS locomotor score compared with the control group at both 7 days and 42 days after SCI (1.4 vs 0.2 points and 4.0 vs 1.4 points, respectively). Phospholipids at 7 days after SCI showed unique distribution patterns. In particular, PCs containing docosahexaenoic acid (DHA) localised in the gray matter region was significantly higher in the MR16-1–treated group than in the control group, at 7 days post-SCI.
Discussion
MR16-1 treatment showed to improve locomotor BMS score after 7 days of SCI compared with that observed in the control group. Spinal cord injury had induced inflammation; injury sites showed changes in the lipid content. We had previously reported that PC containing DHA mostly expressed in neuron cells decrease on injury sites. In this study, we observed that MR16-1 recovered PC containing DHA at the injury site. This may be associated with the recovery of motor function.
