Two-photon-excited fluorescence microscopy as a tool to investigate the efficacy of methylprednisolone in a mouse spinal cord injury model.
METHODS
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).


BACKGROUND
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).


CONCLUSIONS
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.


METHODS
N/A.
*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.
Yiling
Y
Zhang
Lihai
L
Zhang
Jing
J
Shen
Chao
C
Chen
Zhi
Z
Mao
Wei
W
Li
Wen-Biao
WB
Gan
Peifu
P
Tang
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001369:Axons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D004195:Disease Models, Animal; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006614:Hindlimb / Q000294:innervation*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000473:pathology; D008775:Methylprednisolone / Q000494:pharmacology*; D051379:Mice; D008822:Mice, Transgenic; D017628:Microglia / Q000187:drug effects / Q000473:pathology; D036641:Microscopy, Fluorescence, Multiphoton; D009043:Motor Activity / Q000187:drug effects; D020127:Recovery of Function; D013116:Spinal Cord / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D013119:Spinal Cord Injuries / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology / Q000503:physiopathology; D013997:Time Factors
D000893:Anti-Inflammatory Agents; D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D008775:Methylprednisolone