Electroacupuncture stimulates the proliferation and differentiation of endogenous neural stem cells in a rat model of ischemic stroke.

Electroacupuncture (EA) may stimulate neurogenesis in animal models of ischemic stroke; however, the associated mechanisms are not clear. The present study aimed to evaluate the neurogenesis efficacy of EA on ischemic stroke and the underlying associated mechanisms. A model of middle cerebral artery occlusion (MCAO) was employed as the rat model of brain ischemia and reperfusion. EA treatment at the GV20 (Baihui) and GV14 (Dazhui) acupoints was conducted for 30 min daily following MCAO. Immunofluorescence was performed to measure the number of bromodeoxyuridine (BrdU)/nestin- or BrdU/doublecortin (DCX)-positive cells in the sham, MCAO and MCAO + EA groups. Results indicated that EA stimulation significantly decreased the neurological score and neuronal loss in rats in the MCAO group (both P<0.05). Furthermore, immunostaining assays indicated that BrdU/nestin- and BrdU/DCX-positive cells in EA-treated rats were significantly increased (P<0.05) when compared with the rats in the MCAO group, indicating EA may induce the proliferation and differentiation of endogenous neural stem cells (eNSCs) during cerebral ischemia-reperfusion. In addition, EA treatment significantly enhanced the protein expression levels of plasticity-related gene 5 (PRG5), a critical neurogenesis factor, and significantly decreased the protein expression levels of three neurogenesis inhibiting molecules, NogoA, lysophosphatidic acid and RhoA (all P<0.05). These results suggested that EA promotes the proliferation and differentiation of eNSCs, likely through modulating PRG5/RhoA signaling.

Cell permeable HMGB1-binding heptamer peptide ameliorates neurovascular complications associated with thrombolytic therapy in rats with transient ischemic stroke.

BACKGROUND: Blood-brain barrier (BBB) breakdown and inflammatory responses are the major causes of tissue-type plasminogen activator (tPA)-induced hemorrhagic transformation (HT), while high-mobility group box 1 (HMGB1) exacerbates inflammatory damage to BBB during the process of brain ischemia/reperfusion. This study aimed to investigate the change of HMGB1 after thrombolytic therapy and whether blocking HMGB1 could ameliorate the neurovasculature complications secondary to tPA treatment in stroke rats. METHODS: Sera from acute stroke patients and rats with thrombolytic therapy were collected to investigate HMGB1 secretion. Male Sprague-Dawley rats with 2 h or 4.5 h middle cerebral artery occlusion were continuously infused with tPA followed by administration of membrane permeable HMGB1-binding heptamer peptide (HBHP). The mortality rate, neurological score, HT, brain swelling, BBB permeability, and inflammatory factors were determined. RESULTS: The results revealed that HMGB1 levels were elevated in both stroke patients and rats after tPA treatment. Blocking HMGB1 signaling by HBHP in the rat model of 4.5 h brain ischemia significantly attenuated tPA-related complications, including mortality rate, the degree of hemorrhage, brain swelling, neurological deficits, BBB impairment, microglia activation, and the expressions of inflammatory cytokines. CONCLUSIONS: tPA treatment might induce HMGB1 secretion while blocking HMGB1 with HBHP could markedly reduce the risk of thrombolysis-associated brain hemorrhage and mortality through attenuating BBB damage and inflammatory reactions. These results indicate that HMGB1 may potentiate the risk of HT in tPA administration and that blocking HMGB1 signaling would be helpful in preventing complications brought by thrombolysis in ischemic stroke. TRIAL REGISTRATION: http://www.chictr.org.cn . Unique identifier: ChiCTR-OOC-16010052. Registered 30 November 2016.

IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Experimental Stroke Mice.

Background: Innate immune response to neuronal death is one of the key events of the pathogenesis of ischemic brain injury. Interleukin-1 receptor-associated kinase (IRAK)-M, encoded by gene Irak3, negatively regulates toll-like receptor signaling by interacting with the MyD88-IRAK-4-IRAK-1 complex and blocking the phosphorylation and dissociation of IRAK-1. Its function in the ischemic stroke is unknown. Objective: This study aims to investigate whether IRAK-M deficiency could exacerbate neuroinflammation and neurovascular injuries during cerebral ischemia and reperfusion. Methods: Male C57BL/6 mice and Irak3 knockout mice were subjected to 45 min of middle cerebral artery occlusion and 4 or 24 h of reperfusion. Transcription of Irak3 gene was evaluated by quantitative real-time PCR (qRT-PCR). Then, infarct volume, neurological score, brain water content, and Evans blue leakage were compared between knock-out and wild-type mice after reperfusion. Through the observation of gross brain specimen after cerebral ischemia, the incidence of hemorrhage transformation was compared between KO and WT mice. To explore underlying signaling pathways involved in IRAK-M deficiency, major proinflammatory cytokines and NF-κB signaling were measured by qRT-PCR and Western blot. Results: The expression of IRAK-M peaked at 1 h after reperfusion, and then gradually decreased within the first 24 h, which was abolished by blocking the expression of hypoxia induced factor 1α. IRAK-M deficiency increased infarct volume, brain edema, the incidence of hemorrhage transformation, and the permeability of blood-brain barrier. In addition, the NF-κB-mediated expressions of proinflammatory cytokines and the activation of microglia in the ipsilateral brain from knock-out mice were much higher than those in wild-type littermates. Conclusion: IRAK-M deletion exacerbates neurovascular damages which are related to the pronounced activation of NF-κB signaling and neuroinflammatory responses during cerebral ischemia-reperfusion in mice. Our study indicates that IRAK-M has neuroprotective effect and has potential to facilitate the development of new pharmaceuticals that reduce neurovascular complications.

HMGB1 binding heptamer peptide improves survival and ameliorates brain injury in rats after cardiac arrest and cardiopulmonary resuscitation.

Excessive inflammatory response produced after cardiac arrest and cardiopulmonary resuscitation (CA/CPR) is one of major causes of cerebral injury. High mobility group box 1 (HMGB1) is a pro-inflammatory cytokine and its role in brain injury after CA/CPR is unclear. Herein we investigated whether blocking HMGB1 signaling could ease brain injury after CA/CPR. Male Sprague-Dawley rats (n=181) were subjected to 8-min Asphyxia CA model or Sham operation. The ELISA data revealed both resuscitated patients and animals had elevated HMGB1 level in sera, compared with the healthy volunteers or Sham operative rats, respectively (P<0.01). Rats successfully resuscitated from CA were then randomly treated with either membrane permeable (TAT-fused) HMGB1 binding heptamer peptide (HBHP) or Scramble peptide. Results showed that HBHP treatment markedly improved 7-day survival rate, reduced neurological deficit scores, and prevented neuronal and dendrite loss in hippocampal CA1 region. Moreover, HBHP inhibited the activation of microglia and astrocytes and downregulated the mRNA and protein expressions of proinflammatory factors. We finally blocked toll-like receptor-4 (TLR4, one of HMGB1 receptors) with a specific antagonist TAK-242 before CA induction to confirm the detrimental effect of HMGB1 signaling and found blocking TLR4 could also attenuate the neuronal degeneration, as well as reduce NF-κB-mediated inflammatory signaling. Our findings indicate that CA/CPR can induce HMGB1 release to serum, while blocking HMGB1 signaling with peptide may improve the survival and attenuate post-resuscitation brain injury in the rat model of CA/CPR. TLR4 antagonist may also offer neuroprotective effects through weakening HMGB1-mediated proinflammatory reactions.