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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-?B) activity has a similar profile as TLR4 activity. It is consisted with the results of NF-?B mRNA and protein expression changes and activation of downstream cytokine, IL-1?. Expectedly, intrathecal infusion of pyrrolidine dithiocarbamate (PDTC), a NF-?B 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-?B with minocycline or PDTC treatment accordingly perform the same protective effects on I/R injury. CONCLUSION: 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-?B/ IL-1? pathway. To inhibit the positive feedback loop of TLR4-microglia-NF-?B/ IL-1? pathway by minocycline, TAK-242 (TLR4 inhibitor) and pyrrolidine dithiocarbamate (PDTC, NF-?B inhibitor) may provide new targets for treating I/R injury in clinic.

SUBMITTER: Li XQ

PROVIDER: S-EPMC4022154 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Intrathecal antagonism of microglial TLR4 reduces inflammatory damage to blood-spinal cord barrier following ischemia/reperfusion injury in rats.

Li Xiao-Qian XQ Wang Jun J Fang Bo B Tan Wen-Fei WF Ma Hong H

Molecular brain 20140421

<h4>Background</h4>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.<h4>Results</h4>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 sp ...[more]

PMID: 24751148

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Project description:BackgroundSpinal cord ischemia reperfusion (IR) injury causes inflammation and subsequently increases blood-spinal cord barrier leakage and Toll-like receptor 4 (TLR4) pathway activation. MicroRNAs (miRs) effectively regulate numerous target mRNAs during ischemia. However, their roles during IR injury are poorly understood. We investigated miRs involvement, particularly miR-27a, in TLR4 pathway-mediated inflammatory responses after IR.MethodWe used a genomics approach to examine changed miRs of rats that had undergone 14 minutes of ischemia, followed by 24 or 72 hours of reperfusion. Quantitative RT-PCR was used to identify and confirm the miRs involved in regulating TLR4 pathway activation. We scanned miR databases for potential miR targets and confirmed these targets by quantitative RT-PCR. The miR mimic and anti-miR oligonucleotides (AMOs) were intrathecally injected at 12-hour intervals beginning three days before the ischemia. The effects of miRs on the TLR4 pathway and downstream cytokines were analyzed by PCR, western blotting, and ELISA. Double immunofluorescence staining was perfumed to determine the relationship between the targets and TLR4. Blood-spinal cord barrier (BSCB) permeability was examined using Evans blue (EB) dye.ResultsA microarray analysis revealed that at 24 hours post-injury, three miRs were upregulated (>2.0 fold) and 15 miRs were downregulated (<0.5 fold), and at 72 hours, four miRs were upregulated and 14 were downregulated compared to their levels in sham-operated controls. We focused on miR-27a, which is predicted to contain sequences complementary to the 3'-untranslated region (UTR) of Toll-like receptor adaptor molecule 2 (TICAM-2). Double immunostaining indicated that TLR4 activation correlated with changes in TICAM-2 expression. Compared to the rats in the IR and negative control groups, intrathecal infusion of the miR-27a mimic attenuated IR-induced TLR4 activation and inflammatory damage to the BSCB, which was shown as decreased EB extravasation and lower levels of nuclear factor kappa-B (NF-κB) and lnterleukin (IL)-1β at 24 and 72 hours after reperfusion, whereas pretreatment with miR-27a AMO aggravated these injuries.ConclusionsWe present the first evidence that miRs play an important role in spinal cord IR injury. We identified TICAM-2 as a novel target of miR-27a. miR-27a upregulation attenuates IR-induced inflammatory damage to the BSCB by negatively regulating TICAM-2 of the TLR4 signaling pathway and inhibiting the NF-κB/IL-1β pathway. These results provide new therapeutic targets for IR injury treatment.

Project description:Angina pectoris is cardiac pain that is a common clinical symptom often resulting from myocardial ischemia. Spinal cord stimulation (SCS) is effective in treating refractory angina pectoris, but its underlying mechanisms have not been fully elucidated. The spinal dorsal horn is the first region of the central nervous system that receives nociceptive information; it is also the target of SCS. In the spinal cord, glial (astrocytes and microglia) activation is involved in the initiation and persistence of chronic pain. Thus, the present study investigated the possible cardiac pain‑relieving effects of SCS on spinal dorsal horn glia in chronic myocardial ischemia (CMI). CMI was established by left anterior descending artery ligation surgery, which induced significant spontaneous/ongoing cardiac pain behaviors, as measured using the open field test in rats. SCS effectively improved such behaviors as shown by open field and conditioned place preference tests in CMI model rats. SCS suppressed CMI‑induced spinal dorsal horn microglial activation, with downregulation of ionized calcium‑binding adaptor protein‑1 expression. Moreover, SCS inhibited CMI‑induced spinal expression of phosphorylated‑p38 MAPK, which was specifically colocalized with the spinal dorsal horn microglia rather than astrocytes and neurons. Furthermore, SCS could depress spinal neuroinflammation by suppressing CMI‑induced IL‑1β and TNF‑α release. Intrathecal administration of minocycline, a microglial inhibitor, alleviated the cardiac pain behaviors in CMI model rats. In addition, the injection of fractalkine (microglia‑activating factor) partially reversed the SCS‑produced analgesic effects on CMI‑induced cardiac pain. These results indicated that the therapeutic mechanism of SCS on CMI may occur partially through the inhibition of spinal microglial p38 MAPK pathway activation. The present study identified a novel mechanism underlying the SCS‑produced analgesic effects on chronic cardiac pain.

Project description:Purpose To evaluate whether noninvasive molecular imaging technologies targeting myeloperoxidase (MPO) can reveal early inflammation associated with spinal cord injury after thoracic aortic ischemia-reperfusion (TAR) in mice. Materials and Methods The study was approved by the institutional animal care and use committee. C57BL6 mice that were 8-10 weeks old underwent TAR (n = 55) or sham (n = 26) surgery. Magnetic resonance (MR) imaging (n = 6) or single photon emission computed tomography (SPECT)/computed tomography (CT) (n = 15) studies targeting MPO activity were performed after intravenous injection of MPO sensors (bis-5-hydroxytryptamide-tetraazacyclododecane [HT]-diethyneletriaminepentaacetic acid [DTPA]-gadolinium or indium 111-bis-5-HT-DTPA, respectively). Immunohistochemistry and flow cytometry were used to identify myeloid cells and neuronal loss. Proinflammatory cytokines, keratinocyte chemoattractant (KC), and interleukin 6 (IL-6) were measured with enzyme-linked immunosorbent assay. Statistical analyses were performed by using nonparametric tests and the Pearson correlation coefficient. P < .05 was considered to indicate a significant difference. Results Myeloid cells infiltrated into the injured cord at 6 and 24 hours after TAR. MR imaging confirmed the presence of ischemic lesions associated with mild MPO-mediated enhancement in the thoracolumbar spine at 24 hours compared with the sham procedure. SPECT/CT imaging of MPO activity showed marked MPO-sensor retention at 6 hours (P = .003) that continued to increase at 24 hours after TAR (P = .0001). The number of motor neurons decreased substantially at 24 hours after TAR (P < .01), which correlated inversely with in vivo inflammatory changes detected at molecular imaging (r = 0.64, P = .0099). MPO was primarily secreted by neutrophils, followed by lymphocyte antigen 6 complexhigh monocytes and/or macrophages. There were corresponding increased levels of proinflammatory cytokines KC (P = .0001) and IL-6 (P = .0001) that mirrored changes in MPO activity. Conclusion MPO is a suitable imaging biomarker for identifying and tracking inflammatory damage in the spinal cord after TAR in a mouse model. © RSNA, 2016 Online supplemental material is available for this article.

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Intrathecal antagonism of microglial TLR4 reduces inflammatory damage to blood-spinal cord barrier following ischemia/reperfusion injury in rats.

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Intrathecal antagonism of microglial TLR4 reduces inflammatory damage to blood-spinal cord barrier following ischemia/reperfusion injury in rats.

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