Rapamycin suppresses astrocytic and microglial activation and reduces development of neuropathic pain after spinal cord injury in mice.
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1 Rapamycin suppresses astrocytic and microglial activation and reduces development of neuropathic pain after spinal cord injury in mice. Satoshi Tateda, MD, Haruo Kanno, MD, PhD, Hiroshi Ozawa, MD, PhD, Akira Sekiguchi, M.D., Ph.D. Kenichiro Yahata, MD, Seiji Yamaya, MD,! Eiji Itoi, MD, PhD Department of Orthopaedic Surgery, Tohoku University! Graduate School of Medicine, Sendai, Japan
2 The mammalian target of rapamycin (mtor) mtor signaling pathway Growth factors! p-p70s6k PI3K Akt Rheb mtor 4EBP1 Metabolism! Proliferation! Survival! PTEN Rapamycin Atg1 kinase complex Autophagy! Effect of rapamycin Immunosuppression Anti angiogenesis Anti cancer Extended aging Promote autophagy Neuroprotection Neuroregeneration!
3 Background Rapamycin reduces neural tissue damage and locomotor impairment after SCI in mice. (Sekiguchi, J Neurotrauma. 2011) Rapamycin reduces peripheral neuropathic pain in rats (Geranton SM, J Neurosci. 2009) Rapamycin reduces neuropathic pain after SCI?! Objective To examine whether rapamycin reduces neuropathic pain after SCI in mice!
4 SCI model C57BL/6 mouse (Female, weeks) Spinal cord contusion injury :! Modified NYU Impactor! (T10 segment : 10 g rod, height 3 mm) Rapamycin administration Rapamycin-treated group (n=5)! (4 hours after SCI, 1 mg/kg, BW)!! Vehicle-treated group (n=5)!
5 Experimental analysis Behavioral analysis (0-42 days) Locomotor function : Basso mouse scale (BMS) score, subscore Mechanical allodynia :! von Frey test! Thermal allodynia : Hargreaves test! Histological analysis (42 days, L2/3) Microglia : Iba-1 Astrocyte : GFAP Activation of p38 MAPK : p-p38 Double-staining : Iba-1 and p-p38 Quantification of immunoreactivity The box (100 x100 μm) was placed onto areas of the lateral, central and medial superficial dorsal horn of the lumber spinal cord sections. The total pixels of the stained area were expressed as the immunodensity.
6 Locomotor function BMS score! 10! 8! 6! 4! 2! 0! BMS score BMS subscore Rapamycin Rapa! Vehicle! p<0.05 Pre 0! 1! 3! 7! 14!21!28!35!42! Days post-injury! BMS subscore! 12! 10! 8! 6! 4! 2! 0! Rapamycin Rapa! Vehicle SCI! p<0.01 p<0.05 Pre! 1! 3! 7! 14! 21! 28! 35! 42! Days post-injury! Rapamycin significantly improved locomotion
7 Mechanical and Thermal allodynia 50% Withdrawal Threshold (g) 5! 4! 3! 2! 1! 0! Von Frey test Rapa! Rapamycin Vehicle SCI! p<0.05 Pre! 7! 14! 21! 28! 35! 42! Days post-injury! Withdrawal Latency (S) 20! 16! 12! 8! 4! 0! Hargreaves test Rapa! Rapamycin Vehicle! Vehicle p<0.05 Pre! 7! 14! 21! 28! 35! 42! Days post-injury! Rapamycin significantly improved! mechanical and thermal allodynia
8 Histological analysis (42 days, L2/3) Vehicle! Iba-1 Rapamycin Immunodensity! of Iba-1 staining Iba-1 10! 8! 6! 4! 2! 0! Sham! Vehicle! Rapa! p 0.01 p μm Immunodensity! of GFAP staining! Rapamycin significantly suppressed activation of microglia GFAP GFAP 32 Vehicle! Rapamycin 100 μm Sham! Vehicle! Rapa! Activation of astrocytes were relatively reduced by rapamycin
9 Histological analysis (42 days, L2/3) Vehicle! p-p38 Rapamycin Immunodensity! of p-p38 staining p-p μm 25! 20! 15! 10! 5! 0! Sham! Vehicle! Rapa! p 0.01 p 0.05 Rapamycin significantly suppressed activation of p38 MAPK Iba- 1 Iba- 1 p- p38 p- p38 Vehicle! Rapamycin 250μm Rapamycin decreased p-p38 positive cells in microglia
10 Discussion Rapamycin treatment for SCI Previous study Neural tissue damage Locomotor function (Sekiguchi, J Neurotrauma. 2011) This study Mechanical and thermal allodynia Activation of glial cells and p38 MAPK! in the lumbar spinal cord! Rapamycin reduces neuropathic pain after SCI
11 Previous study Therapeutic mechanism Neuroprotective treatment for acute SCI improved neuropathic pain! Rapamycin for SCI in acute phase Neuroprotective effect (Gris, J. Neurosci. 2004) (J. Voda, Neurosci. Res. 2007) (Bao, J Neurotrauma. 2011) (Sekiguchi, J Neurotrauma. 2011) This study Rapamycin reduced neuropathic pain after SCI Neuroprotective effect of rapamycin in acute phase! improved neuropathic pain after SCI
12 Conclusions Rapamycin significantly improved not only locomotor function but also mechanical and thermal allodynia after SCI.! Rapamycin significantly attenuated glial activation in the lumbar spinal cord. Rapamycin treatment has a significant therapeutic potential to reduce neuropathic pain after SCI. Disclosure declaration! None of the authors has any potential conflict of interest
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