title: Intrathecal antagonism of microglial TLR4 reduces inflammatory damage to blood-spinal cord barrier following ischemia/reperfusion injury in rats.
abstract: BACKGROUND
  Inflammatory reaction in blood-spinal cord barrier (BSCB) plays a crucial role in ischemia/reperfusion (I/R) injury. It has been shown that microglia could be activated through Toll-like receptors (TLRs). Therefore, we hypothesize that TLR4 is involved in the microglial activation and BSCB disruption after I/R.
  
  
  RESULTS
  To verify our hypothesis, we analyzed the behavioral data, changes of BSCB permeability, as well as expressions of microglial marker Iba-1 and TLR4 in spinal cord I/R model induced by 14 min aortic occlusion. Double immunostaining reveals that after I/R, Iba-1 immunoreactivity increased gradually 12 h after reperfusion and maintained at a such level throughout 36 h. Such increasing pattern of Iba-1 expression is consistent with the increases in Evan's Blue (EB) extravasation, spinal water content and mechanical allodynia demonstrated by lowed withdrawal threshold to Von Frey filaments. Moreover, double immunostaining suggested that TLR4 was highly expressed in microglia. Intrathecal infusion of minocycline and TAK-242 (TLR4 inhibitor) treatment attenuated I/R-induced allodynia and BSCB leakage. In contrast, LPS induced TLR4 expression aggregated above-mentioned injuries. Furthermore, the nuclear factor-kappa B (NF-κB) activity has a similar profile as TLR4 activity. It is consisted with the results of NF-κB mRNA and protein expression changes and activation of downstream cytokine, IL-1β. Expectedly, intrathecal infusion of pyrrolidine dithiocarbamate (PDTC), a NF-κB inhibitor, showed similar protective effects as minocycline and TAK-242. In addition, our data show that TLR4 closely involved in I/R-induced inflammatory damage induced neuronal apoptosis. Significantly, neutralizing TLR4 function largely reduced neuronal apoptosis determined by NeuN immunoreactivity in ventral gray matter and increased percentage of double-label cells with cleaved caspase3, whereas LPS reversed these effects. Similarly, inhibitions of microglia and NF-κB with minocycline or PDTC treatment accordingly perform the same protective effects on I/R injury.
  
  
  CONCLUSIONS
  The results indicate that compromised BSCB caused by I/R injury lead to spinal microglial activation and TLR4, its membrane-bound receptor, up-regulation, which then initiate neuro-inflammation and neuro-apoptosis via NF-κB/ IL-1β pathway. To inhibit the positive feedback loop of TLR4-microglia-NF-κB/ IL-1β pathway by minocycline, TAK-242 (TLR4 inhibitor) and pyrrolidine dithiocarbamate (PDTC, NF-κB inhibitor) may provide new targets for treating I/R injury in clinic.
authors.forename: Xiao-Qian
authors.initials: XQ
authors.lastname: Li
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Bo
authors.initials: B
authors.lastname: Fang
authors.forename: Wen-Fei
authors.initials: WF
authors.lastname: Tan
authors.affiliation: Department of Anesthesiology, First Affiliated Hospital, China Medical University, Shenyang 110001, Liaoning, China. mahong5466@yahoo.com.
authors.forename: Hong
authors.initials: H
authors.lastname: Ma
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism; D007249:Inflammation / Q000235:genetics / Q000473:pathology*; D053583:Interleukin-1beta / Q000378:metabolism; D008297:Male; D008840:Microfilament Proteins / Q000378:metabolism; D017628:Microglia / Q000378:metabolism* / Q000473:pathology; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D010539:Permeability; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000097:blood* / Q000473:pathology*; D015398:Signal Transduction; D013116:Spinal Cord / Q000378:metabolism* / Q000473:pathology*; D051197:Toll-Like Receptor 4 / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D053583:Interleukin-1beta; D008840:Microfilament Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D051197:Toll-Like Receptor 4
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