Siglec-6 Signaling Uses Src Kinase Tyrosine Phosphorylation and SHP-2 Recruitment.

Preeclampsia is a pregnancy-specific disorder involving placental abnormalities. Elevated placental Sialic acid immunoglobulin-like lectin (Siglec)-6 expression has been correlated with preeclampsia. Siglec-6 is a transmembrane receptor, expressed predominantly by the trophoblast cells in the human placenta. It interacts with sialyl glycans such as sialyl-TN glycans as well as binds leptin. Siglec-6 overexpression has been shown to influence proliferation, apoptosis, and invasion in the trophoblast (BeWo) cell model. However, there is no direct evidence that Siglec-6 plays a role in preeclampsia pathogenesis and its signaling potential is still largely unexplored. Siglec-6 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like motif in its cytoplasmic tail suggesting a signaling function. Site-directed mutagenesis and transfection were employed to create a series of Siglec-6 expressing HTR-8/SVneo trophoblastic cell lines with mutations in specific functional residues to explore the signaling potential of Siglec-6. Co-immunoprecipitation and inhibitory assays were utilized to investigate the association of Src-kinases and SH-2 domain-containing phosphatases with Siglec-6. In this study, we show that Siglec-6 is phosphorylated at ITIM and ITIM-like domains by Src family kinases. Phosphorylation of both ITIM and ITIM-like motifs is essential for the recruitment of phosphatases like Src homology region 2 containing protein tyrosine phosphatase 2 (SHP-2), which has downstream signaling capabilities. These findings suggest Siglec-6 as a signaling molecule in human trophoblasts. Further investigation is warranted to determine which signaling pathways are activated downstream to SHP-2 recruitment and how overexpression of Siglec-6 in preeclamptic placentas impacts pathogenesis.

Onset Time of Spinal Anaesthesia in Pregnant Females in Knee-Chest Position: A Randomized Controlled Study.

OBJECTIVE: To assess the efficacy of knee-chest position in shortening the time of spinal induction in pregnant women undergoing elective cesarean section. We also assessed for any untoward adverse events that might limit their usefulness in real-life clinical scenarios. METHODS: Prospective, randomized controlled study was done in maternity operating room of tertiary care institution in 45 ASA II pregnant women undergoing elective cesarean section under spinal anaesthesia. Patients were randomly assigned to groups S (supine) and K (kneechest position). After performing subarachnoid block (9 mg of 0.5% hyperbaric bupivacaine and 25 µg fentanyl) in the sitting position, women in group K were maintained in the knee-chest position for 60 seconds. Time to attain block height of T6 and maximum sensory blockade, intraoperative hemodynamics, Bromage score, intraoperative fluid, vasopressor requirement, and respiratory parameters were recorded. The newborn was evaluated using Apgar scores at 1 and 5 minutes. RESULTS: Data of 45 patients were analyzed. Time to attain T6 block height (group K=2.1 ± 0.65 minutes, 95% CI: 1.83-2.39; group S=6.4 ± 0.77 minutes, 95% CI: 6.10-6.78) and time to achieve maximum sensory block height were significantly lower in group K (group K=3.2 ± 1.35 minutes, 95% CI: 2.61-3.78; group S=6.6 ± 0.89 min, CI: 6.19-6.98). The degree of motor block was higher in group K than that of group S at 2 minutes (P=.0002), 4 minutes (P < .0001), and 6 minutes (P < .0001), with no difference at 8 minutes. No statistically significant difference was observed in fluids and vasopressors requirement intraoperatively. CONCLUSIONS: This study provides evidence that the onset of adequate surgical anaesthesia for the cesarean section can be hastened by placing the patient in the knee-chest position for a minute after performing the subarachnoid block in the sitting position.

Dysregulation of hypoxia-inducible factor-1α (Hif1α) expression in the Hmox1-deficient placenta.

INTRODUCTION: Severe hypoxia exists in placentas during early pregnancy, with reoxygenation during mid-gestation. Hypoxia-inducible factor-1α (Hif1α), an oxygen sensor, initiates placental vascular development. We have shown that the placental vasculature in Hmox1-deficient (Hmox1+/-, Het) pregnancies is impaired, with morphological defects similar to Hif1α-deficient placentas. MATERIALS AND METHODS: Whole wild-type (WT) and Het mouse placentas were collected at E8.5 (1%-3% O2) and E9.5-15.5 (8%-10% O2). mRNA levels were determined using real-time RT-PCR or PCR arrays and protein levels using Western blot. Bone marrow-derived macrophages (BMDMs) from WT, Het, and Hmox1 knockout (KO) mice, representing different Hmox1 cellular levels, were generated to study the role of Hmox1 on Hif1α 's response to hypoxia-reoxygenation and gestational age-specific placental lysates. RESULTS: Hif1α was expressed in WT and Het placentas throughout gestation, with protein levels peaking at E8.5 and mRNA levels significantly upregulated from E9.5-E13.5, but significantly lower in Het placentas. Genes associated with angiogenesis (Vegfa, Vegfr1, Mmp2, Cxcl12, Angpt1, Nos3), antioxidants (Sod1, Gpx1), and transcription factors (Ap2, Bach1, Nrf2) were significantly different in Het placentas. In response to in vitro hypoxia-reoxygenation and to WT or Het placental lysates, Hif1α transcription was lower in Het and Hmox1 KO BMDMs compared with WT BMDMs. DISCUSSION: These findings suggest that deficiencies in Hmox1 underlie the insufficient placental Hif1α response to hypoxia-reoxygenation during gestation and subsequently impair downstream placental vascular formation. Therefore, a dysregulation of Hif1α expression caused by any genetic defect or environmental influence in early pregnancy could be the root cause of pregnancy disorders.

Absent or Excessive Corpus Luteum Number Is Associated With Altered Maternal Vascular Health in Early Pregnancy.

Identifying modifiable factors that contribute to preeclampsia risk associated with assisted reproduction can improve maternal health. Vascular dysfunction predates clinical presentation of preeclampsia. Therefore, we examined if a nonphysiological hormonal milieu, a modifiable state, affects maternal vascular health in early pregnancy. Blood pressure, endothelial function, circulating endothelial progenitor cell numbers, lipid levels, and corpus luteum (CL) hormones were compared in a prospective cohort of women with infertility history based on number of CL: 0 CL (programmed frozen embryo transfer [FET], N=18); 1 CL (spontaneous conception [N=16] and natural cycle FET [N=12]); or >3 CL associated with in vitro fertilization [N=11]. Women with 0 or >3 CL lacked the drop in mean arterial blood pressure compared with those with 1 CL (both P=0.05). Reactive hyperemia index was impaired in women with 0 CL compared with 1 CL ( P=0.04) while baseline pulse wave amplitude was higher with > 3 CL compared with 1 CL ( P=0.01) or 0 CL ( P=0.01). Comparing only FET cycles, a lower reactive hyperemia index and a higher augmentation index is noted in FETs with suppressed CL compared with FETs in a natural cycle (both P=0.03). The number of angiogenic and nonangiogenic circulating endothelial progenitor cell numbers was lower in the absence of a CL in FETs ( P=0.01 and P=0.03). Vascular health in early pregnancy is altered in women with aberrant numbers of CL (0 or >3) and might represent insufficient cardiovascular adaptation contributing to an increased risk of preeclampsia.

Ultrasound-Guided Midpoint Transverse Process to Pleura Block in Breast Cancer Surgery: A Case Report.

To avoid the safety issues related to thoracic paravertebral blocks, we performed midpoint transverse process to pleura blocks in 3 patients before general anesthesia for modified radical mastectomies. The midpoint transverse process to pleura blocks served as the major component of multimodal analgesia. With ultrasound guidance, 7 mL of a mixture of 0.75% ropivacaine and 2% lidocaine with epinephrine were deposited at T2, T4, and T6 levels. We noted decreased sensation to cold and pinprick from T2 to T8 dermatome level with sparing of axilla and infraclavicular areas. The maximum pain numeric rating scale score (0-10) was 4 out on movement and none had mean 24-hour numeric rating scale >3.

Comparison of caudal epidural block with paravertebral block for renal surgeries in pediatric patients: A prospective randomised, blinded clinical trial.

STUDY OBJECTIVE: This study was undertaken to compare the analgesic efficacy of ultrasound-guided single-shot caudal block with ultrasound-guided single-shot paravertebral block in children undergoing renal surgeries. DESIGN: Randomised, interventional, blinded clinical trial. SETTING: Operating rooms of All India Institute of Medical Sciences, New Delhi, India. PATIENTS: 50 children aged 2-10 years, of ASA status I/II, posted for elective renal surgeries. INTERVENTIONS: The children were randomised into two groups (Group C-caudal block, Group P-paravertebral block). After induction of general anesthesia, single-shot caudal or paravertebral block was performed under ultrasound guidance, with 0.2% ropivacaine with 1:200000 adrenaline. MEASUREMENTS: Time to first rescue analgesia, time to perform blocks, intraoperative and post-operative hemodynamics, post-operative FLACC scores, incidence of complications, parental satisfaction scores were recorded. MAIN RESULTS: Children in Group P had significantly longer duration of analgesia (p < 0.0004) than Group C. Post-operative FLACC scores (p < 0.005) and analgesic requirements (p < 0.0004) were lower in Group P. The mean fentanyl requirement over 24 h in group P was 0.56 ±â€¯0.82 µg/kg, compared to 1.8 ±â€¯1.2 µg/kg in group C. Parents in Group P reported greater satisfaction (p < 0.02). No complications were seen in either of the groups. CONCLUSION: This study showed superior analgesia and parental satisfaction with single-shot paravertebral block in comparison to single-shot caudal block for renal surgeries in children. However, the block performance in children requires adequate expertise and practice.

A novel out plane technique of midpoint transverse process to pleura block in breast surgery: A case report.

Regional anesthetic techniques have gradually revolutionized the perioperative analgesia in breast surgeries. Recently, midpoint transverse process to pleura block has been described and found to provide excellent opioid-sparing analgesia. We performed the block in a novel out-of-plane technique to decrease the patient-needle interaction time and at the same time achieving good analgesia. The immediate postoperative Numeric Pain Rating Scale score was 0/10 both at rest and on movement, and patient reported a score of 5/10 after 12 h, which get subsided with single dose of nonopioid analgesic.

Hypoxic preconditioning enhances neural stem cell transplantation therapy after intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells ameliorates neurological deficits in animals with intracerebral hemorrhage. However, hemoglobin in the host brain environment causes massive grafted cell death and reduces the effectiveness of this approach. Several studies have shown that preconditioning induced by sublethal hypoxia can markedly improve the tolerance of treated subjects to more severe insults. Therefore, we investigated whether hypoxic preconditioning enhances neural stem cell resilience to the hemorrhagic stroke environment and improves therapeutic effects in mice. To assess whether hypoxic preconditioning enhances neural stem cell survival when exposed to hemoglobin, neural stem cells were exposed to 5% hypoxia for 24 hours before exposure to hemoglobin. To study the effectiveness of hypoxic preconditioning on grafted-neural stem cell recovery, neural stem cells subjected to hypoxic preconditioning were grafted into the parenchyma 3 days after intracerebral hemorrhage. Hypoxic preconditioning significantly enhanced viability of the neural stem cells exposed to hemoglobin and increased grafted-cell survival in the intracerebral hemorrhage brain. Hypoxic preconditioning also increased neural stem cell secretion of vascular endothelial growth factor. Finally, transplanted neural stem cells with hypoxic preconditioning exhibited enhanced tissue-protective capability that accelerated behavioral recovery. Our results suggest that hypoxic preconditioning in neural stem cells improves efficacy of stem cell therapy for intracerebral hemorrhage.

Postconditioning mitigates cell death following oxygen and glucose deprivation in PC12 cells and forebrain reperfusion injury in rats.

Postconditioning mitigates ischemia-induced cellular damage via a modified reperfusion procedure. Mitochondrial permeability transition (MPT) is an important pathophysiological change in reperfusion injury. This study explores the role of MPT modulation underlying hypoxic postconditioning (HPoC) in PC12 cells and studies the neuroprotective effects of ischemic postconditioning (IPoC) on rats. Oxygen-glucose deprivation (OGD) was performed for 10 hr on PC12 cells. HPoC was induced by three cycles of 10-min reoxygenation/10-min rehypoxia after OGD. The MPT inhibitor N-methyl-4-isoleucine cyclosporine (NIM811) and the MPT inducer carboxyatractyloside (CATR) were administered to selective groups before OGD. Cellular death was evaluated by flow cytometry and Western blot analysis. JC-1 fluorescence signal was used to estimate the mitochondrial membrane potential (△Ψm ). Transient global cerebral ischemia (tGCI) was induced via the two-vessel occlusion and hypotension method in male Sprague Dawley rats. IPoC was induced by three cycles of 10-sec reperfusion/10-sec reocclusion after index ischemia. HPoC and NIM811 administration attenuated cell death, cytochrome c release, and caspase-3 activity and maintained △Ψm of PC12 cells after OGD. The addition of CATR negated the protection conferred by HPoC. IPoC reduced neuronal degeneration and cytochrome c release and cleaved caspase-9 expression of hippocampal CA1 neurons in rats after tGCI. HPoC protected PC12 cells against OGD by modulating the MPT. IPoC attenuated degeneration of hippocampal neurons after cerebral ischemia.

Transplantation of neural stem cells that overexpress SOD1 enhances amelioration of intracerebral hemorrhage in mice.

Previous studies have shown that intraparenchymal transplantation of neural stem cells (NSCs) ameliorates neurologic deficits in animals with intracerebral hemorrhage (ICH). However, massive grafted cell death after transplantation, possibly caused by a hostile host brain environment, lessens the effectiveness of this approach. We focused on the effect of oxidative stress against grafted NSCs and hypothesized that conferring antioxidant properties to transplanted NSCs may overcome their death and enhance neuroprotection after ICH. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted NSCs and accelerates amelioration of ICH. Neural stem cells that overexpress SOD1 were administered intracerebrally 3 days after ICH in a mouse model. Histologic and behavioral tests were examined after ICH. Copper/zinc-superoxide dismutase overexpression protected the grafted NSCs via a decrease in production of reactive oxygen species. This resulted in an increase in paracrine factors released by the NSCs, and an increase in surviving neurons in the striatum and a reduction in striatal atrophy. In addition, SOD1 overexpression showed progressive improvement in behavioral recovery. Our results suggest that enhanced antioxidative activity in NSCs improves efficacy of stem cell therapy for ICH.

Prevention of JNK phosphorylation as a mechanism for rosiglitazone in neuroprotection after transient cerebral ischemia: activation of dual specificity phosphatase.

Rosiglitazone, a synthetic peroxisome proliferator-activated receptor-γ (PPARγ) agonist, prevents cell death after cerebral ischemia in animal models, but the underlying mechanism has not been clarified. In this study, we examined how rosiglitazone protects neurons against ischemia. Mice treated with rosiglitazone were subjected to 60 minutes of focal ischemia followed by reperfusion. Rosiglitazone reduced infarct volume after ischemia and reperfusion. We show that this neuroprotective effect was reversed with a PPARγ antagonist. Western blot analysis showed a significant increase in expression of phosphorylated stress-activated protein kinases (c-Jun N-terminal kinase (JNK) and p38) in ischemic brain tissue. Rosiglitazone blocked this increase. Furthermore, we observed that rosiglitazone increased expression of the dual-specificity phosphatase 8 (DUSP8) protein and messenger RNA in ischemic brain tissue. Dual-specificity phosphatase 8 is a mitogen-activated protein kinase phosphatase that can dephosphorylate JNK and p38. Another key finding of the present study was that knockdown of DUSP8 in primary cultured cortical neurons that were subjected to oxygen-glucose deprivation diminished rosiglitazone's effect on downregulation of JNK phosphorylation. Thus, rosiglitazone's neuroprotective effect after ischemia is mediated by blocking JNK phosphorylation induced by ischemia via DUSP8 upregulation.

Complement component 3 inhibition by an antioxidant is neuroprotective after cerebral ischemia and reperfusion in mice.

Oxidative stress after stroke is associated with the inflammatory system activation in the brain. The complement cascade, especially the degradation products of complement component 3, is a key inflammatory mediator of cerebral ischemia. We have shown that pro-inflammatory complement component 3 is increased by oxidative stress after ischemic stroke in mice using DNA array. In this study, we investigated whether up-regulation of complement component 3 is directly related to oxidative stress after transient focal cerebral ischemia in mice and oxygen-glucose deprivation in brain cells. Persistent up-regulation of complement component 3 expression was reduced in copper/zinc-superoxide dismutase transgenic mice, and manganese-superoxide dismutase knock-out mice showed highly increased complement component 3 levels after transient focal cerebral ischemia. Antioxidant N-tert-butyl-α-phenylnitrone treatment suppressed complement component 3 expression after transient focal cerebral ischemia. Accumulation of complement component 3 in neurons and microglia was decreased by N-tert-butyl-α-phenylnitrone, which reduced infarct volume and impaired neurological deficiency after cerebral ischemia and reperfusion in mice. Small interfering RNA specific for complement component 3 transfection showed a significant increase in brain cells viability after oxygen-glucose deprivation. Our study suggests that the neuroprotective effect of antioxidants through complement component 3 suppression is a new strategy for potential therapeutic approaches in stroke.

Interleukin 6-preconditioned neural stem cells reduce ischaemic injury in stroke mice.

Transplantation of neural stem cells provides a promising therapy for stroke. Its efficacy, however, might be limited because of massive grafted-cell death after transplantation, and its insufficient capability for tissue repair. Interleukin 6 is a pro-inflammatory cytokine involved in the pathogenesis of various neurological disorders. Paradoxically, interleukin 6 promotes a pro-survival signalling pathway through activation of signal transducer and activator of transcription 3. In this study, we investigated whether cellular reprogramming of neural stem cells with interleukin 6 facilitates the effectiveness of cell transplantation therapy in ischaemic stroke. Neural stem cells harvested from the subventricular zone of foetal mice were preconditioned with interleukin 6 in vitro and transplanted into mouse brains 6 h or 7 days after transient middle cerebral artery occlusion. Interleukin 6 preconditioning protected the grafted neural stem cells from ischaemic reperfusion injury through signal transducer and activator of transcription 3-mediated upregulation of manganese superoxide dismutase, a primary mitochondrial antioxidant enzyme. In addition, interleukin 6 preconditioning induced secretion of vascular endothelial growth factor from the neural stem cells through activation of signal transducer and activator of transcription 3, resulting in promotion of angiogenesis in the ischaemic brain. Furthermore, transplantation of interleukin 6-preconditioned neural stem cells significantly attenuated infarct size and improved neurological performance compared with non-preconditioned neural stem cells. This interleukin 6-induced amelioration of ischaemic insults was abolished by transfecting the neural stem cells with signal transducer and activator of transcription 3 small interfering RNA before transplantation. These results indicate that preconditioning with interleukin 6, which reprograms neural stem cells to tolerate oxidative stress after ischaemic reperfusion injury and to induce angiogenesis through activation of signal transducer and activator of transcription 3, is a simple and beneficial approach for enhancing the effectiveness of cell transplantation therapy in ischaemic stroke.

Involvement of mitogen-activated protein kinase pathways in expression of the water channel protein aquaporin-4 after ischemia in rat cortical astrocytes.

Brain edema after ischemic brain injury is a key determinant of morbidity and mortality. Aquaporin-4 (AQP4) plays an important role in water transport in the central nervous system and is highly expressed in brain astrocytes. However, the AQP4 regulatory mechanisms are poorly understood. In this study, we investigated whether mitogen-activated protein kinases (MAPKs), which are involved in changes in osmolality, might mediate AQP4 expression in models of rat cortical astrocytes after ischemia. Increased levels of AQP4 in primary cultured astrocytes subjected to oxygen-glucose deprivation (OGD) and 2 h of reoxygenation were observed, after which they immediately decreased at 0 h of reoxygenation. Astrocytes exposed to OGD injury had significantly increased phosphorylation of three kinds of MAPKs. Treatment with SB203580, a selective p38 MAPK inhibitor, or SP600125, a selective c-Jun N-terminal kinase inhibitor, significantly attenuated the return of AQP4 to its normal level, and SB203580, but not SP600125, significantly decreased cell death. In an in vivo study, AQP4 expression was upregulated 1-3 days after reperfusion, which was consistent with the time course of p38 phosphorylation and activation, and decreased by the p38 inhibition after transient middle cerebral artery occlusion (MCAO). These results suggest that p38 MAPK may regulate AQP4 expression in cortical astrocytes after ischemic injury.

Neural stem cells genetically modified to overexpress cu/zn-superoxide dismutase enhance amelioration of ischemic stroke in mice.

BACKGROUND AND PURPOSE: The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke. METHODS: NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke. RESULTS: Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified. CONCLUSIONS: Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

Minocycline-preconditioned neural stem cells enhance neuroprotection after ischemic stroke in rats.

Transplantation of neural stem cells (NSCs) offers a novel therapeutic strategy for stroke; however, massive grafted cell death following transplantation, possibly due to a hostile host brain environment, lessens the effectiveness of this approach. Here, we have investigated whether reprogramming NSCs with minocycline, a broadly used antibiotic also known to possess cytoprotective properties, enhances survival of grafted cells and promotes neuroprotection in ischemic stroke. NSCs harvested from the subventricular zone of fetal rats were preconditioned with minocycline in vitro and transplanted into rat brains 6 h after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from days 0-28 after stroke. For in vitro experiments, NSCs were subjected to oxygen-glucose deprivation and reoxygenation. Cell viability and antioxidant gene expression were analyzed. Minocycline preconditioning protected the grafted NSCs from ischemic reperfusion injury via upregulation of Nrf2 and Nrf2-regulated antioxidant genes. Additionally, preconditioning with minocycline induced the NSCs to release paracrine factors, including brain-derived neurotrophic factor, nerve growth factor, glial cell-derived neurotrophic factor, and vascular endothelial growth factor. Moreover, transplantation of the minocycline-preconditioned NSCs significantly attenuated infarct size and improved neurological performance, compared with non-preconditioned NSCs. Minocycline-induced neuroprotection was abolished by transfecting the NSCs with Nrf2-small interfering RNA before transplantation. Thus, preconditioning with minocycline, which reprograms NSCs to tolerate oxidative stress after ischemic reperfusion injury and express higher levels of paracrine factors through Nrf2 up-regulation, is a simple and safe approach to enhance the effectiveness of transplantation therapy in ischemic stroke.

Induction of thioredoxin-interacting protein is mediated by oxidative stress, calcium, and glucose after brain injury in mice.

Oxidative stress and glucose affect the expression of various genes that contribute to both reactive oxygen species generation and antioxidant systems. However, systemic alteration of oxidative stress-related gene expression in normal brains and in brains with a high-glucose status after ischemic-reperfusion has not been explored. Using a polymerase chain reaction array system, we demonstrate that thioredoxin-interacting protein (Txnip) is induced by both oxidative stress and glucose. We found that Txnip mRNA is induced by ischemic-reperfusion injury and that Txnip is located in the cytoplasm of neurons. Moreover, in vitro oxygen-glucose deprivation (OGD) and subsequent reoxygenation without glucose and in vivo administration of 3-nitropropionic acid also promoted an increase in Txnip in a time-dependent manner, indicating that oxidative stress without glucose can induce Txnip expression in the brain. However, calcium channel blockers inhibit induction of Txnip after OGD and reoxygenation. Using the polymerase chain reaction array with ischemic and hyperglycemic-ischemic samples, we confirmed that enhanced expression of Txnip was observed in hyperglycemic-ischemic brains after middle cerebral artery occlusion. Finally, transfection of Txnip small interfering RNA into primary neurons reduced lactate dehydrogenase release after OGD and reoxygenation. This is the first report showing that Txnip expression is induced in neurons after oxidative or glucose stress under either ischemic or hyperglycemic-ischemic conditions, and that Txnip is proapoptotic under these conditions.

Release of mitochondrial apoptogenic factors and cell death are mediated by CK2 and NADPH oxidase.

Activation of the NADPH oxidase subunit, NOX2, and increased oxidative stress are associated with neuronal death after cerebral ischemia and reperfusion. Inhibition of NOX2 by casein kinase 2 (CK2) leads to neuronal survival, but the mechanism is unknown. In this study, we show that in copper/zinc-superoxide dismutase transgenic (SOD1 Tg) mice, degradation of CK2α and CK2α' and dephosphorylation of CK2ß against oxidative stress were markedly reduced compared with wild-type (WT) mice that underwent middle cerebral artery occlusion. Inhibition of CK2 pharmacologically or by ischemic reperfusion facilitated accumulation of poly(ADP-ribose) polymers, the translocation of apoptosis-inducing factor (AIF), and cytochrome c release from mitochondria after ischemic injury. The eventual enhancement of CK2 inhibition under ischemic injury strongly increased 8-hydroxy-2'-deoxyguanosine and phosphorylation of H2A.X. Furthermore, CK2 inhibition by tetrabromocinnamic acid (TBCA) in SOD1 Tg and gp91 knockout (KO) mice after ischemia reperfusion induced less release of AIF and cytochrome c than in TBCA-treated WT mice. Inhibition of CK2 in gp91 KO mice subjected to ischemia reperfusion did not increase brain infarction compared with TBCA-treated WT mice. These results strongly suggest that NOX2 activation releases reactive oxygen species after CK2 inhibition, triggering release of apoptogenic factors from mitochondria and inducing DNA damage after ischemic brain injury.

NADPH oxidase is involved in post-ischemic brain inflammation.

Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is widely expressed in brain tissue including neurons, glia, and endothelia in neurovascular units. It is a major source of oxidants in the post-ischemic brain and significantly contributes to ischemic brain damage. Inflammation occurs after brain ischemia and is known to be associated with post-ischemic oxidative stress. Post-ischemic inflammation also causes progressive brain injury. In this study we investigated the role of NOX2 in post-ischemic cerebral inflammation using a transient middle cerebral artery occlusion model in mice. We demonstrate that mice with NOX2 subunit gp91(phox) knockout (gp91 KO) showed 35-44% less brain infarction at 1 and 3 days of reperfusion compared with wild-type (WT) mice. Minocycline further reduced brain damage in the gp91 KO mice at 3 days of reperfusion. The gp91 KO mice exhibited less severe post-ischemic inflammation in the brain, as evidenced by reduced microglial activation and decreased upregulation of inflammation mediators, including interleukin-1ß (IL-1ß), tumor necrosis factor-α, inducible nitric oxide synthases, CC-chemokine ligand 2, and CC-chemokine ligand 3. Finally, we demonstrated that an intraventricular injection of IL-1ß enhanced ischemia- and reperfusion-mediated brain damage in the WT mice (double the infarction volume), whereas, it failed to aggravate brain infarction in the gp91 KO mice. Taken together, these results demonstrate the involvement of NOX2 in post-ischemic neuroinflammation and that NOX2 inhibition provides neuroprotection against inflammatory cytokine-mediated brain damage.