Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain.

It has been reported that neutrophil extracellular traps (NETs) play important roles in non-infectious diseases. In ischemic stroke, neutrophils infiltrate damaged brain tissue soon after injury and aggravate inflammation. Using a rat permanent MCAO model, we showed citrullinated histone H3+ (CitH3, a marker of NETosis) induction in neutrophils in leptomeninges and in peripheral blood soon after MCAO. Entry of CitH3+ cells occurred through leptomeninges after 6 h of MCAO and these cells were observed in cerebral cortex from 12 h and subsequently in striatum. It is interesting to note that CitH3+ induction began in circulating neutrophils before they migrated to brain parenchyma and they were detected as intact or lysed form. High mobility group box 1 (HMGB1), a danger associated molecular pattern (DAMP) molecule, was accumulated massively in serum after permanent MCAO and plays a critical role in CitH3 inductions in neutrophils in brain parenchyma and in peripheral blood. Both the all-thiol and disulfide types of HMGB1 induced CitH3 via their specific receptors, CXCR4 and TLR4, respectively. Importantly, HMGB1 not only induced NETosis but was included as a part of the extruded NETs, and contribute to NETosis-mediated neuronal death. Therefore, it would appear a vicious cycle exists between neuronal cell death and NETosis and HMGB1 mediates detrimental effects exerted by this cycle. When NETosis was suppressed by a PAD inhibitor in MCAO animals, delayed immune cell infiltrations were markedly suppressed and damages in blood vessels were significantly mitigated. The study shows NETosis with the involvement of HMGB1 as a mediator in a vicious cycle aggravates inflammation and subsequent damage in the ischemic brain.

Noninvasive intranasal drug administration has been noted to allow direct delivery of drugs to the brain. In the present study, the therapeutic efficacy of intranasal small interfering RNA (siRNA) delivery was investigated in the postischemic rat brain. Fluorescein isothiocyanate (FITC)-labeled control siRNA was delivered intranasally in normal adult rats using e-PAM-R, a biodegradable PAMAM dendrimer, as gene carrier. Florescence-tagged siRNA was found in the cytoplasm and processes of neurons and of glial cells in many brain regions, including the hypothalamus, amygdala, cerebral cortex, and striatum, in 1 hour after infusion, and the FITC-fluorescence was continuously detected for at least 12 hours. When siRNA for high mobility group box 1 (HMGB1), which functions as an endogenous danger molecule and aggravates inflammation, was delivered intranasally, the target gene was significantly depleted in many brain regions, including the prefrontal cortex and striatum. More importantly, intranasal delivery of HMGB1 siRNA markedly suppressed infarct volume in the postischemic rat brain (maximal reduction to 42.8 ± 5.6% at 48 hours after 60 minutes middle cerebral artery occlusion (MCAO)) and this protective effect was manifested by recoveries from neurological and behavioral deficits. These results indicate that the intranasal delivery of HMGB1 siRNA offers an efficient means of gene knockdown-mediated therapy in the ischemic brain.