early transplantation of mesenchymal stem cells after spinal cord injury relieves pain...

Early Transplantation of Mesenchymal Stem Cells After Spinal Cord Injury Relieves Pain Hypersensitivity Through

Suppression of Pain-Related Signaling Cascades and Reduced Inflammatory Cell Recruitment

STEM CELLS 2015;33:1902–1914SHUJI WATANABE,a KENZO UCHIDA,a HIDEAKI NAKAJIMA,a HIDEAKI MATSUO,a DAISUKE SUGITA,a AI YOSHIDA,a KAZUYA HONJOH,a WILLIAM E.B. JOHNSON,b HISATOSHI BABAa

aDepartment of Orthopaedics and Rehabilitation Medicine, Faculty of Medical Sciences, University of Fukui, Matsuoka Shimoaizuki, Eiheiji, Fukui, Japan;

bLife and Health Sciences, Aston University, Aston Triangle, Birmingham, United Kingdom

報告人 : 南台科大生物科技碩專班 2 年級謝宗穎

Compassion. Accountability. EffectivenessCompassion. Accountability. Effectiveness

Introduction

• Chronic pain and dysesthesia are important and frequent complaints in patients with spinal cord injury (SCI) with a reported prevalence varying between 27% and 94%

• The SCI Pain Task Force of the IASP broadly classifies SCI pain into nociceptive (musculoskeletal and visceral) and neuropathic (above-level, at-level, and below-level) pain.


Neuropathic pain

• Neuropathic pain has several distinct features: – lesion of nervous tissue,– pain in an area with sensory deficits, – dysesthesia,– allodynia (pain due to a stimulus which does not

normally provoke pain), – hyperalgesia (an increased response to a stimulus which is

normally painful) – abnormal spatial and temporal summation.Several of these features are also seen in SCI painSeveral of these features are also seen in SCI pain


3 broad categories of pain following SCI (depending on the relation of the pain to the site of injury):

• Siddall and colleagues (2000) defined – (1) above-level pain which occurs at dermatomes rostral to

the injury site, – (2) at-level pain which occurs in dermatomes

corresponding to the site of spinal injury– (3) below-level pain which is localized to dermatomes

distal to the injury site


Feng Tao et al. (Stem Cells. 2013 January ; 31(1): 83–91.) reported beneficial effects of cell transplantation therapy in sensory recovery and reduced pain, however, M. Birdsall Abrams et al.(Restorative Neurology and Neuroscience 27 (2009) 307–321) described harmful effects of cell transplants


A massive release of glutamate at the SCI lesion epicenter triggers upregulation of thechemokine CCL21 both at the site of injury and in cell bodies of STT(spinothalamic tract) neurons whose

axons have been exposed to high concentrations of glutamate. CCL21 is then released at remote sitesfrom the injury activating microglia, that contribute to neuronal hyperexcitability in the spinalcord dorsal horn and thalamus. Abnormal amplification and generation of nociceptive signals

at these levels contributes to chronic pain after injury.


SCI leads to persistent activation of intracellular signaling kinases such as ERK 1/2, p38mitogen activated protein kinases (MAPK) and calcium calmodulin kinases (CaMKII) that

lead to persistent activation of CREB, a transcription factor that contributes directly to factorsthat phosphorylated NR1 subunits of the NMDA channel, leading to persistent

hyperexcitability of spinal and supraspinal neurons. This provides the substrate for persistentor neuropathic pain.


Investigate the effects of BMSC transplantation on post-SCI chronic neuropathic pain

• Focus on the effect of BMSC transplants – Microglia and macrophageMicroglia and macrophage– MAPK signaling at the level of the lesion MAPK signaling at the level of the lesion


Contusion SCI model

• 453 adult male C57BL/6N mice , aged 10~12 weeks (27.5+-0.82g) and 20 adult male CAG-EGFP mice, aged 6~8 weeks (19.4+-0.47g)– Enhanced GFP( EGFP) expression was driven by the

cytomegalovirus early enhancer -actin (CAG)transgene.

• Laminectomy at the T9-T10 vertebral level using a surgical microscope and a contusion SCI was produced using the infinite Horizon Impactor with an impact force of 60 kilodynes

• Sham: laminectomy without SCI performed


2.0*105 BMSC in 3 ul of DMEM


Relationship between survival rate of transplanted BMSCs and Time of transplantation

The greatest survived number seen when BMSCs were transplanted at D3


BMSC transplant on D3 post-SCI

Early transplantation of DMSC after SCI increases locomotor function and decreasesEarly transplantation of DMSC after SCI increases locomotor function and decreasesPain hypersensitivity to mechanical and thermal stimulationPain hypersensitivity to mechanical and thermal stimulation


Immunoreactivity and Western blotting for PKC and p-CREB at D14 post-SCI

BMSC transplantation reduced PKCBMSC transplantation reduced PKC and p-CREB protein levels and p-CREB protein levels in the spinal cord dorsal horn neurons in the spinal cord dorsal horn neurons

Immunofluorescent staining and Western blotting for p-p38 MAPK- and p-ERK1/2-positive CD11b cells

macrophage

astrocyte

neuron

BMSC transplantation decreased the upregulation of p-p38 MAPK-BMSC transplantation decreased the upregulation of p-p38 MAPK-or p-ERK1/2- in CD11b positive cells in the spinal cord dorsal horn.or p-ERK1/2- in CD11b positive cells in the spinal cord dorsal horn.


To determine whether BMSC transplantation affects the recruitment of bone marrow-derived cells into the injured spinal cord, the contusive SCI

was reproduced in chimeric mice wherein the hematogenous cells were GFP label

BMSC transplantation inhibited the recruitment of CD11b and GFP-positive BMSC transplantation inhibited the recruitment of CD11b and GFP-positive hematogenous cells to the lesion after SCIhematogenous cells to the lesion after SCI


In control mice, SCI resulted in an observed increase in the presence of cells that were dual labeled with p-p38 MAPK and GFP- or p-ERK1/2 and GFP within the spinal cord dorsal horn.BMSC transplantation not only appeared to prevent the recruitment of GFP-positive bone marrow-derived cells into the spinal cord dorsal column but it also appeared to decrease their coexpression of p-p38 MAPK and p-ERK1/2


Flow cytometry analysis for the p-p38 MAPK and p-ERK1/2 signaling pathway

Hematogenous Hematogenous macrophagemacrophage

Resident Resident microgliamicroglia

BMSC transplantation suppressed the activation of p-p38 MAPK and p-ERK1/2 in BMSC transplantation suppressed the activation of p-p38 MAPK and p-ERK1/2 in bone marrow-derived macrophage and resident microglia after SCI, and prevented bone marrow-derived macrophage and resident microglia after SCI, and prevented recruitment of hematogenous macrophage into the injuried spinal cord.recruitment of hematogenous macrophage into the injuried spinal cord.


BMSC transplantation reduced disruption of the brain-spinal cord barrier


BMSC transplantation moderated the cytokine profile at the lesion site after SCI

BMSC transplantation decreased protein levels of inflammatory cytokines and BMSC transplantation decreased protein levels of inflammatory cytokines and macrophage recruiting chemokine. While increasing that of GM-CSF macrophage recruiting chemokine. While increasing that of GM-CSF


Conclusion

• Demonstrate that early BMSC transplantation – Decreased the activation of MAPK signaling in injured spinal cord – Altered the localized presence of inflammatory mediator, which will

have contribute to a decrease BSCB disruption and reduction in the recruitment of harmful blood-borne macrophage.

– The net effect of the above changes is likely to play a major role in reducing pain hypersensitivity in the BMSC treated SCI animals.

early transplantation of mesenchymal stem cells after spinal cord injury relieves pain...

Documents

level pain

reduced pain

suppression of pain

pain hypersensitivity

allodynia pain

sci pain task force

site of injury

spinal cord injury sci