Prostaglandin E2 and leukotriene C4 levels following different reperfusion periods in rat brain correlated with morphological changes

Prostaglandin E₂ (PGE₂) and leukotriene C₄ (LTC₄) are the metabolites of arachidonic acid (AA) that increase in forebrain following global ischemia and reperfusion. These mediators are highly potent vasoconstrictors of cerebral arteries leading to enhanced vascular permeability that induces the formation of vasogenic edema. In this study, after developing and experimental animal model simulating the concept of ischemic penumbra in the rat, the levels of PGE₂ and LTC₄ produced in the forebrain were measured and the effects of these mediators in short duration and prolonged reperfusion were investigated and then correlated with nueropathological findings. We found statistically significant reduction both in PGE₂ and LTC₄-like activities after just 10 min ischemia (p<0.05, p<0.05). PGE₂-like activity significantly increased in the 4th and 60th min of reperfusion (p<0.05, p<0.05). In the 15th min of reperfusion, PGE₂ was found to be significantly reduced (p<0.005) that may be due to the formation of free oxygen radicals by activation of PG hydroperoxidase reaction that inhibits PGE₂ production in the cylooxygenase pathway. LTs were not significantly increased in any reperfused group. Inhibition of the lipoxygenase pathway of AA metabolism may occur as a result of 15-HPETE (15-hydroperoxyeicosatetraenoic acid) production. Pathologically, edema and degeneration of brain tissue were seen beginning from the 4th min of reperfusion that reached a peak in the 60th min of reperfusion which is in accordance with biochemical changes in the damaged tissue. It is concluded that by preventing the formation of AA metabolites in the early hours of ischemia and reperfusion, it could be possible to increase blood flow in the ischemic penumbra that should limit the infarct area.     

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

Treadmill Pre-Training Ameliorates Brain Edema in Ischemic Stroke via Down-Regulation of Aquaporin-4: An MRI Study in Rats

Objective

Treadmill pre-training can ameliorate blood brain barrier (BBB) dysfunction in ischemia-reperfusion injury, however, its role in ischemic brain edema remains unclear. This study assessed the neuroprotective effects induced by treadmill pre-training, particularly on brain edema in transient middle cerebral artery occluded model.

Methods

Transient middle cerebral artery occlusion to induce stroke was performed on rats after 2 weeks of treadmill pre-training. Magnetic resonance imaging (MRI) was used to evaluate the dynamic impairment of cerebral edema after ischemia-reperfusion injury. In addition, measurements of wet and dry brain weight, Evans Blue assay and Garcia scores were performed to investigate the cerebral water content, BBB permeability and neurologic deficit, respectively. Moreover, during ischemia-reperfusion injury, the expression of Aquaporin 4 (AQP4) was detected using immunofluorescence and Western bloting analyses.

Results

Treadmill pre-training improved the relative apparent diffusion coefficient (rADC) loss in the ipsilateral cortex and striatum at 1 hour and 2.5 hours after cerebral ischemia. In the treadmill pre-training group, T2W1 values of the ipsilateral cortex and striatum increased less at 7.5 hours, 1 day, and 2 days after stroke while the brain water content decreased at 2 days after ischemia. Regarding the BBB permeability, the semi-quantitative amount of contrast agent leakage of treadmill pre-training group significantly decreased. Less Evans Blue exudation was also observed in treadmill pre-training group at 2 days after stroke. In addition, treadmill pre-training mitigated the Garcia score deficits at 2 days after stroke. Immunofluorescence staining and Western blotting results showed a significant decrease in the expression of AQP4 after treadmill ischemia following pre-training.

Conclusions

Treadmill pre-training may reduce cerebral edema and BBB dysfunction during cerebral ischemia/reperfusion injury via the down-regulation of AQP4.     

Effect of Propofol Post-treatment on Blood–Brain Barrier Integrity and Cerebral Edema After Transient Cerebral Ischemia in Rats

Although propofol has been reported to offer neuroprotection against cerebral ischemia injury, its impact on cerebral edema following ischemia is not clear. The objective of this investigation is to evaluate the effects of propofol post-treatment on blood–brain barrier (BBB) integrity and cerebral edema after transient cerebral ischemia and its mechanism of action, focusing on modulation of aquaporins (AQPs), matrix metalloproteinases (MMPs), and hypoxia inducible factor (HIF)-1α. Cerebral ischemia was induced in male Sprague–Dawley rats (n = 78) by occlusion of the right middle cerebral artery for 1 h. For post-treatment with propofol, 1 mg kg^?1 min^?1 of propofol was administered for 1 h from the start of reperfusion. Nineteen rats undergoing sham surgery were also included in the investigation. Edema and BBB integrity were assessed by quantification of cerebral water content and extravasation of Evans blue, respectively, following 24 h of reperfusion. In addition, the expression of AQP-1, AQP-4, MMP-2, and MMP-9 was determined 24 h after reperfusion and the expression of HIF-1α was determined 8 h after reperfusion. Propofol post-treatment significantly reduced cerebral edema (P < 0.05) and BBB disruption (P < 0.05) compared with the saline-treated control. The expression of AQP-1, AQP-4, MMP-2, and MMP-9 at 24 h and of HIF-1α at 8 h following ischemia/reperfusion was significantly suppressed in the propofol post-treatment group (P < 0.05). Propofol post-treatment attenuated cerebral edema after transient cerebral ischemia, in association with reduced expression of AQP-1, AQP-4, MMP-2, and MMP-9. The decreased expression of AQPs and MMPs after propofol post-treatment might result from suppression of HIF-1α expression.     

Tat-indoleamine 2,3-dioxygenase 1 elicits neuroprotective effects on ischemic injury

It is well known that oxidative stress participates in neuronal cell death caused production of reactive oxygen species (ROS). The increased ROS is a major contributor to the development of ischemic injury. Indoleamine 2,3-dioxygenase 1 (IDO-1) is involved in the kynurenine pathway in tryptophan metabolism and plays a role as an anti-oxidant. However, whether IDO-1 would inhibit hippocampal cell death is poorly known. Therefore, we explored the effects of cell permeable Tat-IDO-1 protein against oxidative stress-induced HT-22 cells and in a cerebral ischemia/reperfusion injury model. Transduced Tat-IDO-1 reduced cell death, ROS production, and DNA fragmentation and inhibited mitogen-activated protein kinases (MAPKs) activation in H₂O₂ exposed HT-22 cells. In the cerebral ischemia/reperfusion injury model, Tat-IDO-1 transduced into the brain and passing by means of the blood-brain barrier (BBB) significantly prevented hippocampal neuronal cell death. These results suggest that Tat-IDO-1 may present an alternative strategy to improve from the ischemic injury.     

Involvement of Glial Endothelin/Nitric Oxide in Delayed Neuronal Death of Rat Hippocampus After Transient Forebrain Ischemia

1. We examined time- and cell-type-dependent changes in endothelin (ET)-1-like immunoreactivity, ET receptors binding and nitric oxide (NO) synthase (NOS) activity in CA1 subfields of the hippocampus of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion.2. Microglia aggregated in accord with neuronal death and expressed a high density of ET_B receptors and an intense NOS activity in the damaged CA1 pyramidal cell layer, 7 days after the induced transient forebrain ischemia. The increased NOS activity and ET_B receptor in microglia disappeared 28 days after this transient ischemia.3. In contrast to microglia, astrocytes presented a moderate level of ET-1-like immunoreactivity, ET_B receptors, and NOS activity in all areas of the damaged CA1 subfields, 7 days after the ischemia. These events were further enhanced 28 days after the ischemia.4. In light of these findings, the possibility that the microglial and the astrocytic ET_B/NO system largely contributes to development of the neuronal death and to reconstitution of the damaged neuronal tissue, respectively, in the hippocampus subjected to a transient forebrain ischemia would have to be considered.     

The effects of indomethacin, NDGA, allopurinol and superoxide dismutase on prostaglandin E2 and leukotriene C4 levels after mesenteric ischemia-reperfusion injury

In this study, the changes of arachidonic acid metabolites after an ischemia-reperfusion (I/R) period are investigated. The cyclooxygenase and lipoxygenase metabolites were found to be significantly increased after a 45 min period of ischemia followed by 5 min of reperfusion. Prostaglandin E₂ (PGE₂)- and leukotriene C₄ (LTC₄)-like activities did not change in the ischemic period, but they both increased after reperfusion. A cyclooxygenase inhibitor indomethacin and lipoxygenase inhibitor nordehydroguaretic acid (NDGA) decreased PGE₂- and LTC₄-like activities, respectively, while allopurinol and superoxide dismutase (SOD) decreased both activities.According to our results, it can be assumed that free oxygen radicals are responsible for the elevation of PGE₂- and LTC₄-like activities and both of these arachidonic acid metabolites and free oxygen radicals are the main necrotizing agents in ischemia-reperfusion induced damage.     

Dynamic Expression Pattern of Neuro-oncological Ventral Antigen 1 (Nova1) in the Rat Brain after Focal Cerebral Ischemia/Reperfusion Insults

The present study aimed to evaluate the expression of neuro-oncological ventral antigen 1 (Nova1) in cerebral ischemia/reperfusion (I/R) insults by immunohistochemistry. The focal cerebral I/R model was induced by right middle cerebral artery occlusion (MCAO) for 120 min followed by 1 day, 7 days, and 14 days of reperfusion in Sprague-Dawley (SD) rats. The results showed that Nova1 was expressed in nearly the whole brain, although with higher density in hippocampus, hypothalamus, cingulate cortex, and medial habenular nucleus. The immunoreactivity of Nova1 neurons was increased dramatically, especially on both sides of the hippocampal CA₁ region, after 1 day of reperfusion. A strong response occurred at the ipsilateral CA₁ region between 1 day and 7 days of reperfusion. Likewise, strong compensatory responses of Nova1 expression were observed on the contralateral side of the striate cortex, dentate gyrus, and hypothalamus. Interestingly, more Nova1 neurons were observed to translocate to the dendrites and growth cones of the axons in the hypothalamus on the ischemic side after 7 days of reperfusion. In conclusion, our data suggest that Nova1 might mediate neuronal responsiveness, and its expression might positively correlate with neural repair after I/R insults in the rat brain.     

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.     

Activation of cPLA2, PKC, and ERKs in the Rat Cerebral Cortex During Ischemia/Reperfusion

Release of the excitotoxic amino acid, glutamate, into the extracellular space during ischemia/reperfusion contributes to neuronal injury and death. To gain insights into the signal transduction pathways involved in glutamate release we examined the time course of changes in enzyme levels and activities of cPLA₂, PKC and ERKs in the rat cerebral cortex after four vessel (4VO) ischemia followed by reperfusion. Measurement both by enzymatic assay and Western blot analysis showed significant increases in the activity and protein levels of cPLA₂ during 10–20 min of ischemia. Activity remained elevated at 10 min and 20 min of reperfusion, whereas cPLA levels had returned to base line levels after 20 min of reperfusion. PKC activity increased significantly in the particulate, but not in the cytosolic, fractions both during ischemia and reperfusion. Increases in PKC levels were recorded in the particulate fraction during ischemia and reperfusion, and in the cytosolic fraction during ischemia. Western blot analysis with a phosphospecific antibody for characterization of MAPK (ERKs) activation revealed significantly increased phosphorylation of ERK₁, and ERK₂ in the particulate fraction, of ERK₂ in the cytosolic fraction, during ischemia and of both enzymes in the particulate and cytosolic fractions after 10 min of reperfusion. The relevance of the results to glutamate release is discussed.     

Activation of CRHR1 contributes to cerebral endothelial barrier impairment via cPLA2 phosphorylation in experimental ischemic stroke

The activation of corticotrophin-releasing hormone receptor (CRHR) 1 is implicated in neuronal injury in experimental stroke. However, little is known about the relationship between CRHR1 activation and brain endothelial barrier impairment after ischemia and reperfusion (I/R). Recently we have demonstrated that the activation of extracellular signal-regulated kinase (Erk) 1/2 as well as p38 is required for hydrogen peroxide (H₂O₂)-increased cytosolic phospholipase A₂ (cPLA₂) phosphorylation in bEnd3 cells. Using this in vitro ischemic-like model, we found that both blockade and interference of CRHR1 inhibited H₂O₂-enhancd p38, Erk1/2 and cPLA₂ phosphorylation and in turn suppressed monolayer hyperpermeability and ZO-1 redistribution. Then using the transient middle cerebral artery occlusion (tMCAO) mouse model, we revealed that CRHR1 antagonist NBI27914 pretreatment attenuated cPLA₂ phosphorylation, Evans blue dye (EBD) extravasation, tight junction disruption and mitochondrial cytochrome c release. CRHR1 interference also inhibited cortical vascular hyperpermeability. Furthermore, NBI27914 administration attenuated neurovascular injury. After 30 min MCAO with 7 days reperfusion CRHR1 interference alleviated hippocampal blood-brain barrier (BBB) leakage and improved spatial cognitive dysfunction. Thus, our study demonstrates that during ischemic stroke the activation of endothelial CRHR1 contributes to BBB impairment via cPLA₂ phosphorylation.     

Leukotriene receptor antagonists,LY293111 and ONO-1078, protect neurons from the sPLA2-IB-induced neuronal cell death independently of blocking their receptors

In the ischemic brain, leukotrienes (LTs) are increased and their receptor antagonists protect neurons. However, it has not yet been sufficiently clarified how antagonists for LT receptors exhibit neuroprotective effects. In the present study, we evaluated protective effects of receptor antagonists for LTB₄ (LY293111) and cysteinyl LTs (ONO-1078) in the primary culture of rat cortical neurons. The group IB secretory phospholipase A₂ (sPLA₂-IB)-induced neuronal cell death had been established as the in vitro model for cerebral ischemia. sPLA₂-IB triggered the influx of Ca²⁺ into neurons via L-type voltage-dependent calcium channel (L-VDCC). Subsequently, the enzyme produced eicosanoids including LTB₄ before neuronal cell death. Neither administration of LTB₄ nor cysteinyl LTs such as LTC₄, LTD₄ and LTE₄ killed neurons. However, both LY293111 and ONO-1078 significantly prevented neurons from the neurotoxicity of sPLA₂-IB, suggesting that the two LT receptor blockers protected neurons through alternative pathways beside LT receptors. An L-VDCC blocker does not only inhibit the influx of Ca²⁺ into neurons but also rescues neurons from the sPLA₂-IB-induced neuronal cell death. The two LT receptor antagonists also blocked the sPLA₂-IB-induced Ca²⁺ influx significantly. Thus, LTs exhibited no neurotoxicity, but their receptor antagonists protected neurons directly in the in vitro ischemic model. Furthermore, the suppression of L-VDCC appeared to be involved in the neuroprotective effects of LY293111 and ONO-1078 independent of blocking their receptors.     

Chronic administration of ethyl docosahexaenoate decreases mortality and cerebral edema in ischemic gerbils

Dietary docosahexaenoic acid (DHA) intake can decrease the level of membrane arachidonic acid (AA), which is liberated during cerebral ischemia and implicated in the pathogenesis of brain damage. Therefore, in the present study, we investigated the effects of chronic ethyl docosahexaenoate (E-DHA) administration on mortality and cerebral edema induced by transient forebrain ischemia in gerbils. Male Mongolian gerbils were orally pretreated with either E-DHA (100, 150 mg/kg) or vehicle, once a day, for 4 weeks and were subjected to transient forebrain ischemia by bilateral common carotid occlusion for 30 min. The content of brain lipid AA at the termination of treatment, the survival ratio, change of regional cerebral blood flow (rCBF), brain free AA level, thromboxane B(2) (TXB(2)) production and cerebral edema formation following ischemia and reperfusion were evaluated. E-DHA (150 mg/kg) pretreatment significantly increased survival ratio, prevented post-ischemic hypoperfusion and attenuated cerebral edema after reperfusion compared with vehicle, which was well associated with the reduced levels of AA and TXB(2) in the E-DHA treated brain. These data suggest that the effects of E-DHA pretreatment on ischemic mortality and cerebral edema could be due to reduction of free AA liberation and accumulation, and its metabolite synthesis after ischemia and reperfusion by decreasing the content of membrane AA.     

Calcitonin gene-related peptide prevents blood-brain barrier injury and brain edema induced by focal cerebral ischemia reperfusion

Cerebral ischemia is one of the diseases that most compromise the human species. Therapeutic recovery of blood-brain barrier (BBB) disruption represents a novel promising approach to reduce brain injury after stroke. To determine the effects of calcitonin gene-related peptide (CGRP) on the BBB participate in stroke progression, rat cerebral ischemia reperfusion injury was induced by a 2-hour left transient middle cerebral artery occlusion (MCAO) using an intraluminal filament, followed by 46h of reperfusion. CGRP (1μg/ml) at the dose of 3μg/kg (i.p.) was administered at the beginning of reperfusion. Subsequently, 48h after MCAO, arterial blood pressure, infarct volume, water content, BBB permeability, BBB ultrastructure, levels of aquaporin-4 (AQP4) and its mRNA were evaluated. CGRP could reduce arterial blood pressure (P<0.001), infarct volume (P<0.05), cerebral edema (P<0.01), BBB permeability (P<0.05), AQP4 mRNA expression (P<0.05) and AQP4 protein expression (P<0.01). Furthermore, CGRP treatment improved ultrastructural damage of capillary endothelium cells and decreased the loss of the tight junction observed by transmission electronic microscopy (TEM) after 46h of reperfusion. Our findings show that CGRP significantly reduced postischemic increase of brain edema with a 2-hour therapeutic window in the transient model of focal cerebral ischemia. Moreover, it seems that at least part of the anti-edematous effects of CGRP is due to decrease of BBB disruption by improving ultrastructural damage of capillary endothelium cells, enhancing basal membrane, and inhibiting AQP4 and its mRNA over-expression. The data of the present study provide a new possible approach for acute stroke therapy by administration of CGRP.     

Endothelial cell apoptosis in chronically obstructed and reperfused pulmonary artery

Background

Endothelial dysfunction is a major complication of pulmonary endarterectomy (PTE) that can lead to pulmonary edema and persistent pulmonary hypertension. We hypothesized that endothelial dysfunction is related to increased endothelial-cell (EC) death.

Methods

In piglets, the left pulmonary artery (PA) was ligated to induce lung ischemia then reimplanted into the main PA to reperfuse the lung. Animals sacrificed 5 weeks after ligation (n = 5), 2 days after reperfusion (n = 5), or 5 weeks after reperfusion (n = 5) were compared to a sham-operated group (n = 5). PA vasoreactivity was studied and eNOS assayed. EC apoptosis was assessed by TUNEL in the proximal and distal PA and by caspase-3 activity assay in the proximal PA. Gene expression of pro-apoptotic factors (thrombospondin-1 (Thsp-1) and plasminogen activator inhibitor 1 (PAI-1)) and anti-apoptotic factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) was investigated by QRT-PCR.

Results

Endothelium-dependent relaxation was altered 5 weeks after ligation (p = 0.04). The alterations were exacerbated 2 days after reperfusion (p = 0.002) but recovered within 5 weeks after reperfusion. EC apoptosis was increased 5 weeks after PA ligation (p = 0.02), increased further within 2 days after reperfusion (p < 0.0001), and returned to normal within 5 weeks after reperfusion. Whereas VEGF and bFGF expressions remained unchanged, TSP and PAI-1 expressions peaked 5 weeks after ligation (p = 0.001) and returned to normal within 2 days after reperfusion.

Conclusion

Chronic lung ischemia induces over-expression of pro-apoptotic factors. Lung reperfusion is followed by a dramatic transient increase in EC death that may explain the development of endothelial dysfunction after PE. Anti-apoptotic agents may hold considerable potential for preventing postoperative complications.     

Complex role of STIM1 in the activation of store-independent Orai1/3 channels

Orai proteins contribute to Ca²⁺ entry into cells through both store-dependent, Ca²⁺ release–activated Ca²⁺ (CRAC) channels (Orai1) and store-independent, arachidonic acid (AA)-regulated Ca²⁺ (ARC) and leukotriene C₄ (LTC₄)-regulated Ca²⁺ (LRC) channels (Orai1/3 heteromultimers). Although activated by fundamentally different mechanisms, CRAC channels, like ARC and LRC channels, require stromal interacting molecule 1 (STIM1). The role of endoplasmic reticulum–resident STIM1 (ER-STIM1) in CRAC channel activation is widely accepted. Although ER-STIM1 is necessary and sufficient for LRC channel activation in vascular smooth muscle cells (VSMCs), the minor pool of STIM1 located at the plasma membrane (PM-STIM1) is necessary for ARC channel activation in HEK293 cells. To determine whether ARC and LRC conductances are mediated by the same or different populations of STIM1, Orai1, and Orai3 proteins, we used whole-cell and perforated patch-clamp recording to compare AA- and LTC₄-activated currents in VSMCs and HEK293 cells. We found that both cell types show indistinguishable nonadditive LTC₄- and AA-activated currents that require both Orai1 and Orai3, suggesting that both conductances are mediated by the same channel. Experiments using a nonmetabolizable form of AA or an inhibitor of 5-lipooxygenase suggested that ARC and LRC currents in both cell types could be activated by either LTC₄ or AA, with LTC₄ being more potent. Although PM-STIM1 was required for current activation by LTC₄ and AA under whole-cell patch-clamp recordings in both cell types, ER-STIM1 was sufficient with perforated patch recordings. These results demonstrate that ARC and LRC currents are mediated by the same cellular populations of STIM1, Orai1, and Orai3, and suggest a complex role for both ER-STIM1 and PM-STIM1 in regulating these store-independent Orai1/3 channels.     

Neuronal Damage Using Fluoro-Jade B Histofluorescence and Gliosis in the Gerbil Septum Submitted to Various Durations of Cerebral Ischemia

The extent of neuronal damage/death in some brain regions is highly correlated to duration time of transient ischemia. In the present study, we carried out neuronal degeneration/death and glial changes in the septum 4 days after 5, 10, 15, and 20 min of transient cerebral ischemia using gerbils. To examine neuronal damage, Fluoro-Jade B (F-J B, a marker for neuronal degeneration) histofluorescence staining was used. F-J B positive (⁺) cells were detected in the septo-hippocampal nucleus (SHN) of the septum only in the 20 min ischemia-group; the mean number of F-J B⁺ neurons was 14.9 ± 2.5/400 μm² in a section. Gliosis of astrocytes and microglia was examined using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium-binding adapter molecule 1 (Iba-1), respectively. In all the ischemia-groups, GFAP- and Iba-1-immunoreactive astrocytes and microglia, respectively, were increased in number, and apparently tended to be increased in their immunoreactivity. Especially, in the 20 min ischemia-group, the number and immunoreactivity of Iba-immunoreactive microglia was highest and strongest in the ischemic SHN 4 days after ischemia–reperfusion. In brief, our findings showed that neuronal damage/death in the SHN occurred and gliosis was apparently increased in the 20 min ischemia-group at 4 days after ischemia–reperfusion.     

ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells

The CysLT₂ receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT₂) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT₁, hCysLT₂ and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT₂ receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT₁, pcDNA3.1(+)-hCysLT₂ and pcDNA3.1-rGPR17. Leukotriene C₄ (LTC₄) and LTD₄ were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC₄ and LTD₄ significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT₂, but not in those expressing hCysLT₁ and rGPR17. In hCysLT₂-HEK293 cells, LTC₄ induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT₂ receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC₄-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT₂ receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC₄ and LTD₄.     

Effect of indomethacin on airway contraction and the release of LTC4-like material

Ovalbumin (OA) and arachidonic acid (AA) were used to induce contractions of sensitized guinea-pig tracheal and lung preparations in the presence and absence of indomethacin. Leukotriene (LT)C₄-like material released from these tissues was extracted from the bathing fluid and measured by radioimmunoassay. Challenge with either OA or AA induced release of LTC₄-like material from both parenchyma and trachea, AA inducing a greater release than OA although OA induced greater contractions. This suggested that OA-induced the synthesis of other bronchoconstrictor compounds than LTC₄. Although indomethacin enhanced OA- and AA-induced contractions of trachea, there was no enhancement of the release of LTC₄-like material, suggesting enhancement by indomethacin was a result of the inhibition of the synthesis of prostaglandin E₂ and not diversion of AA into the lipoxygenase pathway. Indomethacin had no effect on OA-induced contractions of parenchyma, but attenuated those induced by AA. Indomethacin had no modulatory effect on the release of LTC₄-like material in the parenchyma. The results demonstrate that indomethacin does not result in increased synthesis of LTs in the airways.