Takayuki Hirai, Kenzo Uchida, Hideaki Nakajima, Tomoo Inukai, Naoto Takeura, Shuji Watanabe, Hisatoshi Baba
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1 Chronic progressive compression induces the phenotype changes of the activated microglia/macrophages in the spinal cord of spinal hyperostotic twy/twy mouse: implications in human cervical compressive myelopathy. Takayuki Hirai, Kenzo Uchida, Hideaki Nakajima, Tomoo Inukai, Naoto Takeura, Shuji Watanabe, Hisatoshi Baba Department of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui
2 BACKGROUND C-OPLL Cervical spondylotic myelopathy C2 The neuroinflammatory processes that are thought to underlie the condition are poorly understood. C6 MRI Baba H, et al., Spine 1999 Uchida K, Baba H, et al., J Neurosurg Spine 2004 Uchida K, Baba H, et al., Spine 2009 Uchida K, Baba H, et al., Eur J Nucl Med Mol Imaging 2012 PET-MRI The present study assessed the localized prevalence of activated M1 and M2 microglia/macrophages in twy/twy mouse that develop spontaneous cervical spinal cord compression, as a model of human disease.
3 % area at Th1 vertebral level ANIMAL MODEL:twy/twy mouse HE staining * 24W spontaneous calcified deposits posteriorly at the C1-C2 vertebral level 12W 18W 24W CT scanning (FOV=50mm, matrix= ) CT ** ** spinal canal (CT) spinal cord (tissue) w Th1 level 24 w 0 12W 18W 24W
4 METHODS Immunofluorescence staining Microglia/macrophages antibody (CD11b) Flow cytometry neurotoxic type (M1 type); inos, CD16/32 neuroprotection type (M2 type); arginase-1, CD206 Double Immunofluorescence staining CD11b stain + anti- inos antibody + anti- CD16/32 antibody + anti- arginase-1 antibody + anti- CD206 antibody Immunoblot analysis Helper T1(Th1) cytokines (IFN-γ, TNF-α, IL-6) Helper T2(Th2) cytokines (IL-4, IL-10, IL13) neurotrophic factors (BDNF, NGF) phagocytic activity (Mac-2) Kigerl KA, Popovich PG, et al., J Neurosci 2009 inos or arginase-1 +anti- BDNF antibody +anti- NGF antibody +anti- Mac-2 antibody
5 RESULT1 -Change of the localization of microglia and neuron Increased prevalence of activated microglia/macrophages after spinal cord compression correlates to neuronal changes.
6 RESULT2 -Active change of microglia/macrophages The resting microglia population decreased in association with increased severity of spinal cord compression.
7 RESULT3 -M1, M2 phenotype CD11b/iNOS M1 type (neurotoxic) CD11b/CD16/32 M2 type (neuroprotection) CD11b/arginase-1 CD11b/CD206 The prevalence of phenotypically activated microglia/macrophages in association with increased severity of spinal cord compression.
8 RESULT4 -phenotype of activated microglia/macrophages M1 type microglia M2 type microglia M1 M2 Expression of the M1 phenotype in activated microglia/macrophages correlated to increased severity of spinal cord compression.
9 RESULT5 -Cytokine expression and phagocytic activity
10 RESULTS6 -neurotrophic factor and phagocytic capacity activity - BDNF NGF Mac-2 inos arginase-1 inos arginase-1 inos arginase-1 Expression of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac)-2 correlated to the phenotype of activated microglia/macrophages and increased spinal cord compression.
11 DISCUSSION Chronic compression Resting microglia Activated microglia/macrophage M1 type M2 type Th1 Cytokines (TNF-α, IL-6,IL-1β) Th2 Cytokines (IL-4, IL-10, IL-13) BDNF, NGF Laskin DL, Chem Res Toxicol 2009 neurotoxic neuroprotection M1 Cell death apoptosis M2 Neuro-glial reserve and survival Chronic and progressive spinal compression induced over-expression of Th1 cytokines M1 type
12 CONCLUSIONS Chronically compressed spinal cord presents enhanced expression of cytokines promoting populations of classically activated macrophages (M1 phenotype); the deleterious effects may be partially responsible for the neuronal loss and demyelination in the pathophysiology of CSM and OPLL, while it also promote the M2 phenotype populations caused by alternative activation.
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