Zhen-Ni Guo

Lipoxin A4 Reduces Inflammation Through Formyl Peptide Receptor 2/p38 MAPK Signaling Pathway in Subarachnoid Hemorrhage Rats

Background and Purpose— Lipoxin A4 (LXA4) has been reported to reduce inflammation in several neurological injury models. We studied the effects of LXA4 on neuroinflammation after subarachnoid hemorrhage (SAH) in a rat model. Methods— Two hundred and thirty-eight Sprague–Dawley male rats, weight 280–320 g, were used. Exogenous LXA4 (0.3 and 1.0 nmol) were injected intracerebroventricularly at 1.5 hours after SAH. Neurological scores, brain water content, and blood–brain barrier were evaluated at 24 hours after SAH; Morris water maze and T-maze tests were examined at 21 days after SAH. The expression of endogenous LXA4 and its receptor formyl peptide receptor 2 (FPR2), as well as p38, interleukin-1&bgr;, and interleukin-6 were studied either by ELISA or by Western blots. Neutrophil infiltration was observed by myeloperoxidase staining. FPR2 siRNA was used to knock down LXA4 receptor. Results— The expression of endogenous LXA4 decreased, and the expression of FPR2 increased after SAH. Exogenous LXA4 decreased brain water content, reduced Evans blue extravasation, and improved neurological functions and improved the learning and memory ability after SAH. LXA4 reduced neutrophil infiltration and phosphorylation of p38, interleukin-1&bgr;, and interleukin-6. These effects of LXA4 were abolished by FPR2 siRNA. Conclusions— Exogenous LXA4 inhibited inflammation by activating FPR2 and inhibiting p38 after SAH. LXA4 may serve as an alternative treatment to relieve early brain injury after SAH.

Macrophage-Inducible C-Type Lectin/Spleen Tyrosine Kinase Signaling Pathway Contributes to Neuroinflammation After Subarachnoid Hemorrhage in Rats

Background and Purpose— Macrophage-inducible C-type lectin (Mincle, CLEC4E) receptor is reported involved in neuroinflammation in cerebral ischemia and traumatic brain injury. This study was designed to investigate the role of Mincle and its downstream spleen tyrosine kinase (Syk) signal pathway in early brain injury after subarachnoid hemorrhage (SAH) in a rat model. Methods— Two hundred fifteen male Sprague-Dawley rats (280–320 g) were subjected to endovascular perforation model of SAH. SAH grade, neurological score, and brain water content were measured at 24 hours after SAH. Mincle/Syk, as well as CARD9 (a member of the caspase-associated recruitment domain [CARD], involved in innate immune response), interleukin-1&bgr;,and myeloperoxidase expressions were analyzed by Western blot at 24 hours after SAH. Specific cell types that expressed Mincle were detected with double immunofluorescence staining. Mincle small interfering RNA, recombinant SAP130, and a selective Syk phosphorylation inhibitor piceatannol were used for intervention. Results— Brain water content increased and neurological functions decreased in rats after SAH. The expression of SAP130, Mincle, Syk, and p-Syk increased at 12 hours and peaked at 24 hours after SAH. Mincle small interfering RNA reduced interleukin-1&bgr; and infiltration of myeloperoxidase positive cells, decreased brain water content, and improved neurological functions at 24 hours after SAH. Recombinant SAP130 upregulated the expression of p-Syk and CARD9 and increased the levels of interleukin-1&bgr; and myeloperoxidase, even though it did not increase brain water content nor it deteriorated neurological function at 24 hours after SAH. Syk inhibitor piceatannol reduced brain edema at 24 hours after SAH. Conclusion— Mincle/Syk is involved in early brain injury after SAH, and they may serve as new targets for therapeutic intervention.

Peroxisome Proliferator–Activated Receptor β/δ Alleviates Early Brain Injury After Subarachnoid Hemorrhage in Rats

Background and Purpose— Early brain injury is proposed to be the primary cause of the poor outcome after subarachnoid hemorrhage (SAH), which is closely related to the neural apoptosis. To date, the relationship between peroxisome proliferator–activated receptor &bgr;/&dgr; (PPAR&bgr;/&dgr;) and nuclear factor-&kgr;B/matrix metalloproteinase-9 (NF-&kgr;B/MMP-9) pathway, both of which are closely related to apoptotic effects, has been poorly studied in SAH. The present study was undertaken to evaluate the effects of PPAR&bgr;/&dgr; on early brain injury and NF-&kgr;B/MMP-9 pathway after SAH in rats. Methods— SAH model was established by injecting nonheparinized autologous arterial blood into the prechiasmatic cistern in male Sprague–Dawley rats. Adenoviruses or small interfering RNAs were injected into the right lateral cerebral ventricle to, respectively, up- or downregulate PPAR&bgr;/&dgr; expression before SAH. All animals were assessed with a neurological score and then killed at 24 hours after SAH surgery. The indexes of brain water content, blood–brain barrier permeability, and apoptosis were used to detect brain injury. The expression of PPAR&bgr;/&dgr;, NF-&kgr;B, and MMP-9 were measured by immunohistochemistry, gelatin zymography, and Western Blot methods, respectively. In addition, GW0742, a specific agonist of PPAR&bgr;/&dgr;, was used to treat SAH in rats, the effects of which were evaluated by neurological scoring and Evans blue extravasation. Results— Overexpression of PPAR&bgr;/&dgr; by adenoviruses treatment significantly ameliorated brain injury with improvement in neurological deficits, brain edema, blood–brain barrier impairment, and neural cell apoptosis at 24 hours after SAH in rats, whereas downregulation of PPAR&bgr;/&dgr; by small interfering RNAs administration resulted in the reverse effects of the above. The expression levels of NF-&kgr;B and MMP-9 were markedly downregulated when PPAR&bgr;/&dgr; increased after PPAR&bgr;/&dgr; adenovirus transfection and upregulated when PPAR&bgr;/&dgr; decreased by PPAR&bgr;/&dgr; small interfering RNAs treatment. Moreover, GW0742 improved neurological deficits and reduced Evans blue extravasation at 24 hours after SAH. Conclusions— PPAR&bgr;/&dgr;’s overexpression may attenuate early brain injury after rats’ SAH administration, which reduces neural apoptosis possibly through blocking NF-&kgr;B/MMP-9 pathway.

Role of PDGF-D and PDGFR-β in neuroinflammation in experimental ICH mice model.

OBJECTIVE Inflammation plays a key role in the pathophysiological processes after intracerebral hemorrhage (ICH). Post-ICH macrophages infiltrate the brain and release pro-inflammatory factors (tumor necrosis factor-α), amplifying microglial activation and neutrophil infiltration. Platelet-derived growth factor receptor-β (PDGFR-β) is expressed on macrophages and its activation induces the recruitment of macrophages. Platelet-derived growth factor-D (PDGF-D) is an agonist with a significantly higher affinity to the PDGFR-β compared to another isoform of the receptor. In this study, we investigated the role of PDGF-D in the pro-inflammatory response after ICH in mice. METHODS A blood injection model of ICH was used in eight-week old male CD1 mice (weight 30g). Some mice received an injection of plasmin or PDGF-D. Gleevec, a PDGFR inhibitor, was administered at 1, 3 or 6h post-ICH. Plasmin was administered with or without PDGF-D siRNAs mixture or scramble siRNA. A plasmin-antagonist, ε-Aminocaproic acid (EACA), was co-administrated with the blood. The effects of ICH and treatment on the brain injury and post-ICH inflammation were investigated. RESULTS ICH resulted in the overexpression of PDGF-D, associated with the infiltration of macrophages. PDGFR-inhibition decreased ICH-induced brain injury, attenuating macrophage and neutrophil infiltration, reducing microglial activation and TNF-α production. Administration of recombinant PDGF-D induced TNF-α production, and PDGFR-inhibition attenuated it. A plasmin-antagonist suppressed PDGFR-β activation and microglial activation. Plasmin increased PDGF-D expression, and PDGF-D inhibition reduced neutrophil infiltration. CONCLUSION ICH-induced PDGF-D accumulation contributed to post-ICH inflammation via PDGFR activation and enhanced macrophage infiltration. The inhibition of PDGFR had an anti-inflammatory effect. Plasmin is a possible upstream effector of PDGF-D. The targeting of PDGF-D may provide a novel way to decrease brain injury after ICH.

Hyperbaric Oxygen Preconditioning Attenuates Hemorrhagic Transformation Through Reactive Oxygen Species/Thioredoxin-Interacting Protein/Nod-Like Receptor Protein 3 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats.

Objectives:To clarify whether hyperbaric oxygen preconditioning can attenuate hyperglycemia-enhanced hemorrhagic transformation and to establish a role for Nod-like receptor protein 3 inflammasome in the pathophysiology of hemorrhagic transformation. Design:Controlled prospective animal study. Setting:University research laboratory. Subjects:Male Sprague-Dawley rats weighing 260–280 g. Interventions:Rats received 1-hour-long hyperbaric oxygen preconditioning for five consecutive days. Hyperglycemic middle cerebral artery occlusion model was induced at 24 hours after the last hyperbaric oxygen exposure. Reactive oxygen species scavenger (N-acetyl-L-cysteine), thioredoxin-interacting protein small interfering RNA, and Nod-like receptor protein 3 small interfering RNA were given in different groups separately to verify the possible pathway. Measurements and Main Results:Rats were randomly divided into sham, middle cerebral artery occlusion, middle cerebral artery occlusion + dextrose, middle cerebral artery occlusion + dextrose + normobaric oxygen preconditioning, middle cerebral artery occlusion + dextrose + hyperbaric oxygen, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + N-acetyl-L-cysteine, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + control small interfering RNA, middle cerebral artery occlusion + dextrose + hyperbaric oxygen + thioredoxin-interacting protein small interfering RNA, and middle cerebral artery occlusion + dextrose + hyperbaric oxygen + Nod-like receptor protein 3 small interfering RNA groups. Hyperglycemia was induced by administration of 50% dextrose (6 mL/kg) intraperitoneally 30 minutes before middle cerebral artery occlusion. Control small interfering RNA/thioredoxin-interacting protein small interfering RNA or Nod-like receptor protein 3 small interfering RNA (500 pmol/5 &mgr;L) were injected intracerebroventricularly 72 hours before middle cerebral artery occlusion for intervention. The neurologic scores, infarction and hemorrhage volumes, the expression of Nod-like receptor protein 3, and its downstream targets were analyzed. Hyperbaric oxygen preconditioning decreased both infarction and hemorrhage volumes and improved neurobehavioral function. In addition, hyperbaric oxygen preconditioning provided additional protective effects in hemorrhagic transformation, which was independent of infarction volume. The benefits of hyperbaric oxygen preconditioning on hyperglycemic middle cerebral artery occlusion rats were reversed after blocking the reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway. Conclusions:Nod-like receptor protein 3 inflammasome played an important role in hyperglycemia-enhanced hemorrhagic transformation. Hyperbaric oxygen preconditioning attenuated hemorrhagic transformation through reactive oxygen species/thioredoxin-interacting protein/Nod-like receptor protein 3 pathway.

Angiopoietin-like 4: A double-edged sword in atherosclerosis and ischemic stroke?

Ischemic stroke is one of the leading causes of death in the world, and thus is a major public health concern. Atherosclerosis, also known as atherogenesis, is a crucial risk factor for cerebral ischemia, yet how it develops remains largely unknown. It has been found, however, that angiopoietin-like protein 4 (ANGPTL4), a protein expressed in vascular endothelial cells, plays a role in the pathophysiology of atherosclerosis and may therefore be involved in ischemic stroke. ANGPTL4 activity is associated with endothelial cell integrity, inflammation, oxidative stress, and lipid metabolism. ANGPTL4 also serves as a potent inhibitor of the lipoprotein lipase, and may inhibit atherogenesis via regulating inflammatory signaling and lipid metabolism. In addition, ANGPTL4 plays a role in the regulation of oxidative stress. However, there currently exists a controversy on the role of ANGPTL4 in endothelial cells. Some studies indicate that ANGPTL4 can protect the integrity of endothelial cells, while others have shown that it can be destructive to the endothelium, thereby leading to the initiation of atherosclerosis. Thus, the effects of ANGPTL4 on development of atherosclerosis and thereby ischemic stroke, are undefined. Further research is needed to better understand ANGPTL4-mediated signaling pathways in endothelial function and to determine its potentials as therapeutic target for atherosclerosis and ischemic stroke.

Recombinant Gas6 augments Axl and facilitates immune restoration in an intracerebral hemorrhage mouse model

Axl, a tyrosine kinase receptor, was recently identified as an essential component regulating innate immune response. Suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 are potent Axl-inducible negative inflammatory regulators. This study investigated the role of Axl signaling pathway in immune restoration in an autologous blood-injection mouse model of intracerebral hemorrhage. Recombinant growth arrest-specific 6 (Gas6) and R428 were administrated as specific agonist and antagonist. In vivo knockdown of Axl or suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 by siRNA was applied. After intracerebral hemorrhage, the expression of endogenous Axl, soluble Axl, and Gas6 was increased, whereas the expression of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 was inhibited. Recombinant growth arrest-specific 6 administration alleviated brain edema and improved neurobehavioral performances. Moreover, enhanced Axl phosphorylation with cleavage of soluble Axl (sAxl), and an upregulation of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 were observed. In vivo knockdown of Axl and R428 administration both abolished the effect of recombinant growth arrest-specific 6 on brain edema and also decreased the expression suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3. In vivo knockdown of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3 aggravated cytokine releasing despite of recombinant growth arrest-specific 6. In conclusion, Axl plays essential role in immune restoration after intracerebral hemorrhage. And recombinant growth arrest-specific 6 attenuated brain injury after intracerebral hemorrhage, probably by enhancing Axl phosphorylation and production of suppressor of cytokine signaling 1 and suppressor of cytokine signaling 3.

Hyperbaric oxygen therapy for traumatic brain injury: bench-to-bedside

Traumatic brain injury (TBI) is a serious public health problem in the United States. Survivors of TBI are often left with significant cognitive, behavioral, and communicative disabilities. So far there is no effective treatment/intervention in the daily clinical practice for TBI patients. The protective effects of hyperbaric oxygen therapy (HBOT) have been proved in stroke; however, its efficiency in TBI remains controversial. In this review, we will summarize the results of HBOT in experimental and clinical TBI, elaborate the mechanisms, and bring out our current understanding and opinions for future studies.

Hyperbaric Oxygen Reduces Infarction Volume and Hemorrhagic Transformation Through ATP/NAD(+)/Sirt1 Pathway in Hyperglycemic Middle Cerebral Artery Occlusion Rats.

Background and Purpose— Energy depletion is a critical factor leading to cell death and brain dysfunction after ischemic stroke. In this study, we investigated whether energy depletion is involved in hyperglycemia-induced hemorrhagic transformation after ischemic stroke and determined the pathway underlying the beneficial effects of hyperbaric oxygen (HBO). Methods— After 2-hour middle cerebral artery occlusion, hyperglycemia was induced by injecting 50% dextrose (6 mL/kg) intraperitoneally at the onset of reperfusion. Immediately after it, rats were exposed to HBO at 2 atmospheres absolutes for 1 hour. ATP synthase inhibitor oligomycin A, nicotinamide phosphoribosyl transferase inhibitor FK866, or silent mating type information regulation 2 homolog 1 siRNA was administrated for interventions. Infarct volume, hemorrhagic volume, and neurobehavioral deficits were recorded; the level of blood glucose, ATP, and nicotinamide adenine dinucleotide and the activity of nicotinamide phosphoribosyl transferase were monitored; the expression of silent mating type information regulation 2 homolog 1, acetylated p53, acetylated nuclear factor-&kgr;B, and cleaved caspase 3 were detected by Western blots; and the activity of matrix metalloproteinase-9 was assayed by zymography. Results— Hyperglycemia deteriorated energy metabolism and reduced the level of ATP and nicotinamide adenine dinucleotide and exaggerated hemorrhagic transformation, blood–brain barrier disruption, and neurological deficits after middle cerebral artery occlusion. HBO treatment increased the levels of the ATP and nicotinamide adenine dinucleotide and consequently increased silent mating type information regulation 2 homolog 1, resulting in attenuation of hemorrhagic transformation, brain infarction, as well as improvement of neurological function in hyperglycemic middle cerebral artery occlusion rats. Conclusions— HBO induced activation of ATP/nicotinamide adenine dinucleotide/silent mating type information regulation 2 homolog 1 pathway and protected blood–brain barrier in hyperglycemic middle cerebral artery occlusion rats. HBO might be promising approach for treatment of acute ischemic stroke patients, especially patients with diabetes mellitus or treated with r-tPA (recombinant tissue-type plasminogen activator).

Hyperbaric oxygen preconditioning: a reliable option for neuroprotection

Brain injury is the leading cause of death and disability worldwide and clinically there is no effective therapy for neuroprotection. Hyperbaric oxygen preconditioning (HBO-PC) has been experimentally demonstrated to be neuroprotective in several models and has shown efficiency in patients undergoing on-pump coronary artery bypass graft (CABG) surgery. Compared with other preconditioning stimuli, HBO is benign and has clinically translational potential. In this review, we will summarize the results in experimental brain injury and clinical studies, elaborate the mechanisms of HBO-PC, and discuss regimes and opinions for future interventions in acute brain injury.