Premium
Simultaneous knockdown of toll‐like receptors 2 and 4 attenuates the severity of post‐stroke inflammation and brain damage
Author(s) -
Nalamolu Koteswara Rao,
Chelluboina Bharath,
Smith Nathan J,
Klopfenstein Jeffrey D,
Pinson David M,
Wang David Z,
Veeravalli Krishna Kumar
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb507
Subject(s) - tlr2 , tlr4 , neuroprotection , stroke (engine) , inflammation , downregulation and upregulation , gene knockdown , medicine , receptor , pharmacology , brain damage , ischemia , anesthesia , immunology , biology , apoptosis , gene , biochemistry , mechanical engineering , engineering
Toll‐like receptor 2 (TLR2) and TLR4 belong to a family of highly conserved pattern recognition receptors and are well‐known upstream sensors of signaling pathways of innate immunity. Evidence suggests that the induction of TLR2 and TLR4 by endogenous molecules after ischemic stroke contributes to the brain damage. Also, TLR2 and TLR4 upregulation in stroke patients is associated with poor stroke outcome. Recent reports indicated that the genetic deletion of TLR2 or TLR4 is neuroprotective in stroke‐induced animals, but few models have sought to demonstrate this neuroprotection in a therapeutically relevant manner. Therefore, we hypothesize that the pharmacological suppression of TLR2 and TLR4 alone and in combination in stroke‐induced wild‐type animals attenuates the severity of post‐stroke inflammation and brain damage. Male Sprague‐Dawley rats were obtained and randomly assigned to various groups. Rats were subjected to a two‐hour middle cerebral artery occlusion procedure followed by reperfusion. To achieve the specific knockdown of TLR2 and/or TLR4, plasmids expressing shRNAs specific to TLR2 or TLR4 formulated as nanoparticles were administered immediately after reperfusion to various groups of rats intravenously via tail vein at a dose of 1 mg/kg body weight. Untreated and treated stroke‐induced rats along with controls were sacrificed at various post‐reperfusion time points. We performed various techniques such as TTC staining, real time PCR, immunoblot and immunofluorescence analysis in order to investigate the severity of post‐stroke inflammation and brain injury. Our results demonstrated huge upregulation of TLR2 and TLR4 in the ischemic brains of stroke‐induced rats. The expression of TLR2 and TLR4 downstream signaling molecules including the proinflammatory mediators such as IL‐1β, IL‐6, and TNFα was increased until seven days post‐reperfusion, the maximum post‐reperfusion time point tested. shRNA‐mediated TLR2 or TLR4 suppression significantly attenuated the infarct size. Additionally, the extent of ischemic brain injury is significantly inhibited with the simultaneous suppression of TLR2 and TLR4 as compared to their individual treatments. Based on our results, we conclude that the reduction of severity of TLR2‐ and TLR4‐mediated post‐stroke inflammation attenuates ischemic brain damage and can be achieved using shRNA targeting techniques.