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Two-Photon-Excited Fluorescence Microscopy as a Tool to Investigate the Efficacy of Methylprednisolone in a Mouse Spinal Cord Injury Model

Zhang, Yiling MD*,†; Zhang, Lihai MD*; Shen, Jing MD*; Chen, Chao MD*,†; Mao, Zhi MD*; Li, Wei PhD; Gan, Wen-Biao PhD†,‡; Tang, Peifu MD*

doi: 10.1097/BRS.0000000000000218
Basic Science

Study Design. Basic imaging experiment.

Objective. To explore the use of 2-photon–excited fluorescence (2PEF) microscopy to investigate the therapeutic effect of methylprednisolone (MP) in mice with spinal cord injury (SCI).

Summary of Background Data. MP can alleviate secondary SCI through its anti-inflammatory effect; however, how MP regulates axonal dynamics in a compression SCI model is not well characterized. We used 2PEF microscopy to trace axonal dynamics in vivo during MP therapy.

Methods. Two types of transgenic mice (weighing 23–25 g) including YFP-H line (n = 18) and CX3CR1-GFP (n = 18) were used for experimental procedure. Each type of mouse was randomly divided into 3 groups, and the sample size of every subgroup was 6. The sham groups including YFP-H line group (n = 6) and CX3CR1-GFP group (n = 6) received laminectomy only (group 1). SCI groups received saline treatment (group 2) and SCI groups received MP treatment (group 3). Hind limb motor function was evaluated using the Basso Mouse Scale. 2PEF microscopy was used to image in vivo axonal dynamics at baseline and at 0.5 hours, 24 hours, 48 hours, and 72 hours postinjury. Histology was employed to examine pathological changes and microglial/macrophage proliferation after all imaging sessions.

Results. Group 1 exhibited no significant differences in hind limb motor function before versus after surgery. The Basso Mouse Scale scores were significantly lower in groups 2 and 3 than in group 1 (P < 0.05). Degree of recovery was higher in group 3 than in group 2 at 7 days postinjury (P < 0.05). The axons in group 1 remained intact at all time points. The survival rate of axons in groups 2 and 3 progressively decreased at 48 hours postinjury; at 72 hours postinjury, the axon survival rate was higher in group 3 than group 2 (P < 0.05). Histology revealed that group 3 presented milder damage in injured spinal cord than group 2. Microglial/macrophage proliferation was lower in group 3 than in group 2 (P < 0.05).

Conclusion. 2PEF microscopy is useful for detecting early changes, indicating axonal disruption in compression SCI. MP therapy may help alleviate axonal progressive damage and reduce the proliferation of microglia/macrophages in acute SCI.

Level of Evidence: N/A

Two-photon–excited fluorescence microscopy was used to investigate the therapeutic effect of methylprednisolone in compression spinal cord injury. Methylprednisolone therapy showed neuroprotective effect in alleviating axonal progressive damage and reducing the proliferation of microglia/macrophages in compression spinal cord injury model.

*Department of Orthopaedics, the General Hospital of Chinese People's Liberation Army, Beijing, P.R. China

School of Chemical Biology and Biotechnology, Shenzhen Graduate School, Peking University, Shenzhen, P.R. China; and

Molecular Neurobiology Program, Department of Physiology and Skirball Institute, New York University School of Medicine, New York, NY.

Address correspondence and reprint requests to Peifu Tang, MD, Department of Orthopaedics, the General Hospital of Chinese People's Liberation Army, Beijing 100853, P.R. China; E-mail: tangpeifu301@gmail.com or Wen-Biao Gan, PhD, Molecular Neurobiology Program, Department of Physiology and Skirball Institute, New York University School of Medicine, New York, NY 10016, E-mail: Wenbiao.Gan@med.nyu.edu

Acknowledgment date: October 28, 2013. First revision date: December 4, 2013. Second revision date: January 2, 2014. Acceptance date: January 6, 2014.

The device(s)/drug(s) is/are FDA approved or approved by corresponding national agency for this indication.

No funds were received in support of this work.

No relevant financial activities outside the submitted work.

© 2014 by Lippincott Williams & Wilkins