Toll-like receptor 4 signaling is involved in PACAP-induced neuroprotection in BV2 microglial cells under OGD/reoxygenation.

OBJECT: The neuroprotective effects of pituitary adenylate cyclise-activating polypeptide (PACAP) have been well documented in vivo and in vitro. However, the mechanisms by which PACAP protected microglia from ischemic/hypoxic injury via inhibition of microglia activation remain unclear. Toll-like receptor 4 (TLR4) plays a considerable role in the induction of innate immune and inflammatory responses. The purpose of this study is to investigate the effect of PACAP on the oxygen and glucose deprivation (OGD)/reoxygenation BV2 microglia and to explore the role of TLR4/myeloid differentiation protein 88 (MyD88)/nuclear factor-kappa B (NF-kappaB) pathway in the neuroprotective effects of PACAP. METHODS: We conducted OGD/reoxygenation by placing BV2 microglia into an airtight chamber and in glucose-free medium. BV2 microglia cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] assay. Western blot was utilized to detect TLR4, MyD88 expression, inhibitory protein of NF-kappaB (IkappaB) phosphorylation/degradation, NF-kappaB activation. Level of tumor necrosis factor-alpha (TNF-alpha) in culture medium was measured with enzyme-linked immunosorbent assay (ELISA). Apoptosis was determined by flow cytometry. RESULTS: We found that pretreatment with PACAP to BV2 cells immediately before OGD/reoxygenation significantly alleviated microglia hypoxic injury. PACAP inhibited upregulation of TLR4, MyD88 and NF-kappaB in BV2 microglial cells exposed to OGD/reoxygenation. PACAP administration also significantly reduced the production of proinflammatory cytokines and apoptosis in BV2 microglia exposed to OGD/reoxygenation. DISCUSSION: Pretreatment with PACAP inhibited activation of the TLR4/MyD88/NF-kappaB signaling pathway and decreased inflammatory cytokine levels, as well as apoptosis in microglia, thereby attenuating microglia hypoxic injury. Our results suggested that TLR4-mediated MyD88-dependent signaling pathway contributed to neuroprotection of PACAP to microglia against OGD/reoxygenation.

Exogenous administration of PACAP alleviates traumatic brain injury in rats through a mechanism involving the TLR4/MyD88/NF-κB pathway.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is effective in reducing axonal damage associated with traumatic brain injury (TBI), and has immunomodulatory properties. Toll-like receptor 4 (TLR4) is an important mediator of the innate immune response. It significantly contributes to neuroinflammation induced by brain injury. However, it remains unknown whether exogenous PACAP can modulate TBI through the TLR4/adapter protein myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway. In this study, we investigated the potential neuroprotective mechanisms of PACAP pretreatment in a weight-drop model of TBI. PACAP38 was microinjected intracerebroventricularly before TBI. Brain samples were extracted from the pericontusional area in the cortex and hippocampus. We found that TBI induced significant upregulation of TLR4, with peak expression occurring 24 h post-trauma, and that pretreatment with PACAP significantly improved motor and cognitive dysfunction, attenuated neuronal apoptosis, and decreased brain edema. Pretreatment with PACAP inhibited upregulation of TLR4 and its downstream signaling molecules MyD88, p-IκB, and NF-κB, and suppressed increases in the levels of the downstream inflammatory agents interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), in the brain tissue around the injured cortex and in the hippocampus. Administration of PACAP both in vitro and in vivo attenuated the ability of the TLR4 agonist lipopolysaccharide (LPS) to increase TLR4 protein levels. Therefore, PACAP exerts a neuroprotective effect in this rat model of TBI, by inhibiting a secondary inflammatory response mediated by the TLR4/MyD88/NF-κB signaling pathway in microglia and neurons, thereby reducing neuronal death and improving the outcome following TBI.

Synergistic myoprotection of L-arginine and adenosine in a canine model of global myocardial ischaemic reperfusion injury.

BACKGROUND: Endogenous nitric oxide and adenosine increase simultaneously to keep the balance of energy demand and supply when the oxygen supply is insufficient, which suggests that nitric oxide and adenosine might exert a synergistic myoprotection during tissue hypoxia. In this study, we tested this hypothesis utilizing a canine model of prolonged global myocardial ischaemic reperfusion injury. METHODS: In this double blind, controlled study, the hearts of 24 anaesthetized mongrel dogs were arrested for 2 hours with aortic cross clamping and blood cardioplegia. The treatment groups were those supplemented with 2 mmol/L L-arginine (ARG), supplemented with 1 mmol/L adenosine (ADO), ARG + ADO supplemented with both, and no supplementation (control) (n = 6 in each group). Haemodynamics, biochemical indices, adenosine triphosphate (ATP) content and myeloperoxidase activities of myocardium were determined to evaluate myocardial injury. Statistical comparison was performed by two way ANOVA. RESULTS: Although the requirements for inotropic supports were higher, the cardiac outputs were lower in control group than in ARG, ADO and the combination groups. Plasma cardiac troponin I levels were higher and the areas of hydropic changes were larger in control group than in ARG and ADO groups. Combination of arginine and adenosine provided further myoprotection with respect to better cardiac performance, lower release of cardiac troponin I, and smaller areas of hydropic changes compared with ARG and ADO groups. ATP content was higher, but myeloperoxidase activities of myocardium were significantly lower in the combination group than in control, ARG and ADO groups (P < 0.05). CONCLUSIONS: Combination of L-arginine and adenosine provides synergistic myoprotection in a canine model of global myocardial ischaemia. Thus, the combination is recommended when the heart is exposed to a prolonged ischaemia during cardiac surgery.

[The in vitro model of inhalation anesthesia using rodent ventilator].

OBJECTIVE: To set up a method of applying inhalation anesthesia in rodent using rodent ventilator and to study the dynamic procedure of the in vitro model. METHODS: The output port of the anesthesia machine was connected to the input port of the rodent ventilator, which was connected to a syringe simulating the lung. After supply of anesthetic gas, the gas samples from the input port of the ventilator and the syringe in the end-expiratory phase were collected at 10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds respectively and were determined using the gas chromatography(GC). The ratios of the anesthetic concentrations of the syringe to that of the input port were calculated (CE/CI). In elimination phase, the gas samples from the syringe were collected at 0,10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds respectively and were determined by GC. The ratios of the anesthetic concentrations of the gas at 10, 20, 30, 40, 50, 60, 90, 120, 180, 300, 600 and 900 seconds to that at 0 second were calculated(C'E/C0). RESULTS: CE/CI increased in the inhalation phase, there was an inverse relationship between CE/CI and time, the correlation coefficients were 0.90, 0.95 and 0.93 respectively (P < 0.01). The mathematical fitting equations were y = -0.19 + 0.19x(-1), y = -7.75 + 0.99x(-1), and y = -7.21 + 0.97x(-1) respectively. C'E/C0 decreased in the elimination phase,the correlation coefficients were 0.90, 0.94 and 0.95 respectively (P < 0.01). The mathematic fitting eqations were y = 5.65-0.02x(-1), y = 7.82-0.01x(-1),and y = 8.20-0.01x(-1), respectively. CONCLUSION: The in vitro model of rodent inhalation anesthesia using the rodent ventilator was set up. The establishment of this model has provided a basis for studies on inhalation anesthesia in rodents.