Targeting Neuronal GPR65 With Delayed BTB09089 Treatment Improves Neurorehabilitation Following Ischemic Stroke.

BACKGROUND: GPR65 (G protein-coupled receptor 65) can sense extracellular acidic environment to regulate pathophysiological processes. Pretreatment with the GPR65 agonist BTB09089 has been proven to produce neuroprotection in acute ischemic stroke. However, whether delayed BTB09089 treatment and neuronal GPR65 activation promote neurorestoration remains unknown. METHODS: Ischemic stroke was induced in wild-type (WT) or GPR65 knockout (GPR65-/-) mice by photothrombotic ischemia. Male mice were injected intraperitoneally with BTB09089 every other day at days 3, 7, or 14 poststroke. AAV-Syn-GPR65 (adenoassociated virus-synapsin-GPR65) was utilized to overexpress GPR65 in the peri-infarct cortical neurons of GPR65-/- and WT mice. Motor function was monitored by grid-walk and cylinder tests. The neurorestorative effects of BTB09089 were observed by immunohistochemistry, Golgi-Cox staining, and Western blotting. RESULTS: BTB09089 significantly promoted motor outcomes in WT but not in GPR65-/- mice, even when BTB09089 was delayed for 3 to 7 days. BTB09089 inhibited the activation of microglia and glial scar progression in WT but not in GPR65-/- mice. Meanwhile, BTB09089 reduced the decrease in neuronal density in WT mice, but this benefit was abolished in GPR65-/- mice and reemerged by overexpressing GPR65 in peri-infarct cortical neurons. Furthermore, BTB09089 increased the GAP43 (growth-associated protein-43) and synaptophysin puncta density, dendritic spine density, dendritic branch length, and dendritic complexity by overexpressing GPR65 in the peri-infarct cortical neurons of GPR65-/- mice, which was accompanied by increased levels of p-CREB (phosphorylated cAMP-responsive element-binding protein). In addition, the therapeutic window of BTB09089 was extended to day 14 by overexpressing GPR65 in the peri-infarct cortical neurons of WT mice. CONCLUSIONS: Our findings indicated that delayed BTB09089 treatment improved neurological functional recovery and brain tissue repair poststroke through activating neuronal GRP65. GPR65 overexpression may be a potential strategy to expand the therapeutic time window of GPR65 agonists for neurorehabilitation after ischemic stroke.

Levosimendan Relaxes Thoracic Aortic Smooth Muscle in Mice by Inhibiting PKC and Activating Inwardly Rectifying Potassium Channels.

ABSTRACT: Studies have examined the therapeutic effect of levosimendan on cardiovascular diseases such as heart failure, perioperative cardiac surgery, and septic shock, but the specific mechanism in mice remains largely unknown. This study aimed to investigate the relaxation mechanism of levosimendan in the thoracic aorta smooth muscle of mice. Levosimendan-induced relaxation of isolated thoracic aortic rings that were precontracted with norepinephrine (NE) or KCl was recorded in an endothelium-independent manner. Vasodilatation by levosimendan was not associated with the production of the endothelial relaxation factors NO and PGI2. The voltage-dependent K+ channel (KV) blocker (4-aminopyridine) and selective KCa blocker (tetraethylammonium) had no effect on thoracic aortas treated with levosimendan, indicating that KV and KCa channels may not be involved in the levosimendan-induced relaxation mechanism. Although the inwardly rectifying K+ channel (Kir) blocker (barium chloride) and the KATP channel blocker (glibenclamide) significantly inhibited levosimendan-induced vasodilation in the isolated thoracic aorta, barium chloride had a much stronger inhibitory effect on levosimendan-induced vasodilation than glibenclamide, suggesting that levosimendan-induced vasodilation may be mediated by Kir channels. The vasodilation effect and expression of Kir 2.1 induced by levosimendan were further enhanced by the PKC inhibitor staurosporine. Extracellular calcium influx was inhibited by levosimendan without affecting intracellular Ca2+ levels in the isolated thoracic aorta. These results suggest that Kir channels play a more important role than KATP channels in regulating vascular tone in larger arteries and that the activity of the Kir channel is enhanced by the PKC pathway.

Diurnal Characteristics of the Orexin System Genes and Its Effects on Pathology at Early Stage in 3xTg-AD Mice.

Orexin and its receptors are closely related to the pathogenesis of Alzheimer's disease (AD). Although the expression of orexin system genes under physiological condition has circadian rhythm, the diurnal characteristics of orexin system genes, and its potential role in the pathogenesis in AD are unknown. In the present study, we hope to elucidate the diurnal characteristics of orexin system genes at the early stage of AD, and to investigate its potential role in the development of AD neuropathology. We firstly detected the mRNA levels of orexin system genes, AD risk genes and core clock genes (CCGs) in hypothalamus and hippocampus in 6-month-old male 3xTg-AD mice and C57BL/6J (wild type, WT) control mice, then analyzed diurnal expression profiles of all genes using JTK_CYCLE algorithm, and did the correlation analysis between expression of orexin system genes and AD risk genes or CCGs. In addition, the expression of ß-amyloid protein (Aß) and phosphorylated tau (p-tau) protein were measured. The results showed that the diurnal mRNA expression profiles of PPO, OX1R, OX2R, Bace2, Bmal1, Per1, Per2 and Cry1 in the hypothalamus, and gene expression of OX1R, OX2R, Bace1, Bmal1, Per1 and Cry2 in the hippocampus in 3xTg-AD mice were different from that in WT mice. Furthermore, there is positive correlation between orexin system genes and AD risk genes or CCGs in the brain in 3xTg-AD mice. In addition, the expression of Aß and p-tau in hippocampus in 3xTg-AD mice were significantly increased, and the expression of p-tau is higher in night than in day. These results indicate that the abnormal expression profiles of orexin system genes and its interaction with AD risk genes or CCGs might exert important role in the pathogenesis of AD, which will increase the expression of Aß and p-tau, and accelerate the development of AD.

Delayed CO2 postconditioning promotes neurological recovery after cryogenic traumatic brain injury by downregulating IRF7 expression.

AIMS: Few treatments are available in the subacute phase of traumatic brain injury (TBI) except rehabilitation training. We previously reported that transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects against cerebral ischemia/reperfusion injury. In this study, it was hypothesized that delayed CO2 postconditioning (DCPC) starting at the subacute phase may promote neurological recovery of TBI. METHODS: Using a cryogenic TBI (cTBI) model, mice received DCPC daily by inhaling 5%/10%/20% CO2 for various time-courses (one/two/three cycles of 10-min inhalation/10-min break) at Days 3-7, 3-14 or 7-18 after cTBI. Beam walking and gait tests were used to assess the effect of DCPC. Lesion size, expression of GAP-43 and synaptophysin, amoeboid microglia number and glia scar area were detected. Transcriptome and recombinant interferon regulatory factor 7 (Irf7) adeno-associated virus were applied to investigate the molecular mechanisms. RESULTS: DCPC significantly promoted recovery of motor function in a concentration and time-course dependent manner with a wide therapeutic time window of at least 7 days after cTBI. The beneficial effects of DCPC were blocked by intracerebroventricular injection of NaHCO3 . DCPC also increased puncta density of GAP-43 and synaptophysin, and reduced amoeboid microglia number and glial scar formation in the cortex surrounding the lesion. Transcriptome analysis showed many inflammation-related genes and pathways were altered by DCPC, and Irf7 was a hub gene, while overexpression of IRF7 blocked the motor function improvement of DCPC. CONCLUSIONS: We first showed that DCPC promoted functional recovery and brain tissue repair, which opens a new therapeutic time window of postconditioning for TBI. Inhibition of IRF7 is a key molecular mechanism for the beneficial effects of DCPC, and IRF7 may be a potential therapeutic target for rehabilitation after TBI.

Delayed Chronic Acidic Postconditioning Improves Poststroke Motor Functional Recovery and Brain Tissue Repair by Activating Proton-Sensing TDAG8.

Acidic postconditioning by transient CO2 inhalation applied within minutes after reperfusion has neuroprotective effects in the acute phase of stroke. However, the effects of delayed chronic acidic postconditioning (DCAPC) initiated during the subacute phase of stroke or other acute brain injuries are unknown. Mice received daily DCAPC by inhaling 5%/10%/20% CO2 for various durations (three cycles of 10- or 20-min CO2 inhalation/10-min break) at days 3-7, 7-21, or 3-21 after photothrombotic stroke. Grid-walk, cylinder, and gait tests were used to assess motor function. DCAPC with all CO2 concentrations significantly promoted motor functional recovery, even when DCAPC was delayed for 3-7 days. DCAPC enhanced the puncta density of GAP-43 (a marker of axon growth and regeneration) and synaptophysin (a marker of synaptogenesis) and reduced the amoeboid microglia number, glial scar thickness and mRNA expression of CD16 and CD32 (markers of proinflammatory M1 microglia) compared with those of the stroke group. Cerebral blood flow (CBF) increased in response to DCAPC. Furthermore, the mRNA expression of TDAG8 (a proton-activated G-protein-coupled receptor) was increased during the subacute phase of stroke, while DCAPC effects were blocked by systemic knockout of TDAG8, except for those on CBF. DCAPC reproduced the benefits by re-expressing TDAG8 in the peri-infarct cortex of TDAG8-/- mice infected with HBAAV2/9-CMV-TDAG8-3flag-ZsGreen. Taken together, we first showed that DCAPC promoted functional recovery and brain tissue repair after stroke with a wide therapeutic time window of at least 7 days after stroke. Brain-derived TDAG8 is a direct target of DCAPC that induces neuroreparative effects.

A1/A2 astrocytes in central nervous system injuries and diseases: Angels or devils?

Astrocytes play a pivotal role in maintaining the central nervous system (CNS) homeostasis and function. In response to CNS injuries and diseases, reactive astrocytes are triggered. By purifying and genetically profiling reactive astrocytes, it has been now found that astrocytes can be activated into two polarization states: the neurotoxic or pro-inflammatory phenotype (A1) and the neuroprotective or anti-inflammatory phenotype (A2). Although the simple dichotomy of the A1/A2 phenotypes does not reflect the wide range of astrocytic phenotypes, it facilitates our understanding of the reactive state of astrocytes in various CNS disorders. This article reviews the recent evidences regarding A1/A2 astrocytes, including (a) the specific markers and morphological characteristics, (b) the effects of A1/A2 astrocytes on the neurovascular unit, and (c) the molecular mechanisms involved in the phenotypic switch of astrocytes. Although many questions remain, a deeper understanding of different phenotypic astrocytes will eventually help us to explore effective strategies for neurological disorders by targeting astrocytes.

Delayed metformin treatment improves functional recovery following traumatic brain injury via central AMPK-dependent brain tissue repair.

Accumulating evidence suggests that chronic metformin posttreatment offers potent neuroreparative effects against acute brain injury. However, in previous studies, metformin was not initially administered beyond 24 h postinjury, and the effects of delayed metformin treatment in traumatic brain injury (TBI) and other types of acute brain injury and the related mechanisms are unclear. To test this, male C57BL/6 mice received once daily metformin treatment (20, 50 or 100 mg/kg/d, i.p.) at day 1-14, day 1-2, day 1-10, day 3-10, day 5-12 or day 5-28 after cryogenic TBI (cTBI). The results showed that 100 mg/kg/d metformin administered at day 1-14 postinjury significantly promoted motor functional recovery in the beam walking and gait tests and reduced the infarct volume. Metformin (100 mg/kg/d) administered at day 1-10 or day 3-10 but not day 1-2 or day 5-12 after cTBI significantly improved motor functional outcomes at day 7 and 14, and reduced the infarct volume at day 14. Interestingly, the therapeutic time window was further expanded when the duration of metformin treatment starting at day 5 postinjury was extended to 2 weeks. Furthermore, compared with cTBI, the administration of metformin at day 3-10 or day 5-28 after cTBI significantly elevated the expression of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) and growth associated protein 43 (an axonal regeneration marker) and the number of vascular branch points and decreased the area of glial scar and the number of amoeboid microglia in the peri-infarct area at day 14 or 28 postinjury. The above beneficial effects of metformin were blocked by the intracerebroventricular injection of the AMPK inhibitor compound C (40 µg/mouse/d). Our data provide the first evidence that metformin has a wide therapeutic time window for at least 5 days after cTBI, during which it can improve functional recovery by promoting tissue repair and inhibiting glial scar formation and microglial activation in a central AMPK-dependent manner.

Hydrophobin-enhanced stability, dispersions and release of curcumin nanoparticles in water.

Most chemotherapeutic drugs commonly suffer from low aqueous solubility that can potentially limit drugs absorption. Drug nanomerization is an advanced approach to overcoming their poor water-solubility. In this study, class I hydrophobin recombinant HGFI (rHGFI)-based curcumin (Cur) nanoparticles (rHGFI-Cur) were prepared by freeze-drying method. The rHGFI-Cur nanocomposites were characterized by contact angle, transmission electron microscopy, fluorescence microscopy and dynamic light scattering. The results showed that rHGFI could lead to the wettability conversion and stability improved of Cur in water. X-ray photoelectron spectroscopy and Fourier transform infrared suggested that rHGFI could non-covalently bind to Cur to render them hydrophilic through hydrophobic forces. Additionally, drug release and cytotoxicity assays illustrated that rHGFI-Cur nanoparticles could facilitate Cur release and exhibited higher cytotoxicity than free Cur for human esophageal cancer cells TE-1. Thus, it suggested that rHGFI has a great potential application for hydrophobic drug delivery without toxicity.[Formula: see text].

In vitro and in vivo release of diclofenac sodium-loaded sodium alginate/carboxymethyl chitosan-ZnO hydrogel beads.

To control release of drugs sensitive to gastrointestinal (GI) environmental effects or irritating to stomach, such as diclofenac sodium (DS), sodium alginate (SA) hydrogel beads are gaining considerable attention gradually. However, due to high swelling ratio, the sustained release performance of SA hydrogel is still far from satisfactory. The objective of this research was to develop new drug delivery device based on SA and ZnO nanoparticles (ZnO NPs). ZnO NPs were prepared by direct precipitation method, and carboxymethyl chitosan (CMCS) acted as stabilizing agent to dominate the preparation of ZnO NPs. The incorporation of CMCS-ZnO NPs resulted in slower and sustained release of DS in vitro. In vivo pharmacokinetics studies showed the bioavailability of DS was better after oral administration of DS-loaded SA/CMCS-ZnO hydrogel beads. These results suggested that SA/CMCS-ZnO hydrogel beads will be a prospective material for loading drugs sensitive to GI environmental effects or irritating to stomach.

Preparation and characterization of multilayer films composed of chitosan, sodium alginate and carboxymethyl chitosan-ZnO nanoparticles.

To deal with serious environmental pollution resulting from plastic packaging materials, biodegradable films using chitosan (CS) are gaining considerable increase gradually. However, chitosan films lack important properties to meet the preserved demands. This study aimed to develop new bio-based films incorporated with carboxymethyl chitosan-ZnO (CMCS-ZnO) nanoparticles and sodium alginate (SA) to overcome the weakness of CS films. CMCS-ZnO nanoparticles were successfully synthesized in the matrix of CMCS through direct precipitation method, which showed an average diameter of 100 nm. Multilayer films with CS film as the outer layer and SA film as the inner layer were prepared by solution casting method. The addition of CMCS-ZnO nanoparticles led to enhanced tensile strength, and to better water vapor resistance. The as-prepared films exhibited distinctive antibacterial activity against S. aureus and E. coli. The results suggested that the as-prepared film is expected to be a promising material for food packaging.

Characterization, release, and antioxidant activity of curcumin-loaded sodium alginate/ZnO hydrogel beads.

In this study, we fabricated a series of novel sodium alginate/ZnO hydrogel beads to optimize the release profile of curcumin (Cur) and to avoid the burst release associated with pure hydrogels, which were used to mitigate the weaknesses of Cur, such as rapid physiological clearance and sensitivity to ultraviolet (UV) light and alkaline solutions. The results show that the composite hydrogel beads exhibit good pH sensitivity and controlled-release capacity, which could prolong the residence time of Cur in the gastrointestinal tract. After exposure to UV irradiation for 6 h, the 1,1-Diphenyl-2-picrylhydrazyl (DPPH) scavenging capacity of Cur-loaded hydrogel beads was decreased by only 13.70%, whereas that of pure Cur decreased by 62.04% under the same conditions; therefore, the encapsulated Cur showed a higher antioxidant activity. The composite hydrogel beads protected the Cur from light degradation and can therefore prolong the antioxidant activity of Cur. These results are beneficial for the design of delivery systems to entrap and control the release of unstable drugs.

Effects of long-term rapamycin treatment on glial scar formation after cryogenic traumatic brain injury in mice.

Glial scar impedes axon regeneration and functional recovery following traumatic brain injury (TBI). Although it has been shown that rapamycin (a specific inhibitor of mammalian target of rapamycin) can reduce astrocyte reactivation in the early stage of TBI, its effect on glial scar formation has not been characterized in TBI and other acute brain injury models. To test this, ICR mice received daily administration of rapamycin (0.5 or 1.5 mg/kg, i.p.) beginning at 1 h after cryogenic TBI (cTBI). The results showed that at 3 d post-injury, 1.5 mg/kg rapamycin increased cTBI-induced motor functional deficits and infarct size, and attenuated astrocyte reactivation in the ipsilateral cortex, while 0.5 mg/kg rapamycin did not worsen brain damage and only slightly attenuated astrocyte reactivation. Furthermore, at 7 and 14 d after cTBI, 0.5 mg/kg rapamycin group showed a better motor functional performance than cTBI group. At 14 d post-injury, 0.5 mg/kg rapamycin significantly reduced the area and thickness of glial scar and chondroitin sulfate proteoglycan expression, accompanied by decreased expression of p-S6 and enhanced expression of growth associated protein 43 (an axon regeneration marker) in the region of glial scar. Our data suggest that long-term treatment with rapamycin can inhibit glial scar formation after cTBI, which may be involved in the mechanisms of increased axon regeneration and improved neurological functional recovery, and low-dose rapamycin may be more beneficial for such a therapy.

Postconditioning-induced neuroprotection, mechanisms and applications in cerebral ischemia.

Ischemic postconditioning (PostC) is defined as a series of rapid intermittent interruptions of blood flow at the phase of reperfusion, which produces neuroprotection against cerebral ischemia/reperfusion injury via mobilizing the brain's own endogenous adaptive mechanisms. Now the concept of conventional ischemic PostC has been extended to limb remote ischemic PostC and chemical PostC with hypoxia, volatile anesthetic, CO2, etc. According to the different temporal profile of PostC, it is divided into rapid and delayed PostC. Rapid PostC is applied within a few seconds to minutes after reperfusion, while delayed PostC is applied at a few hours to days after reperfusion. Although the neuroprotective mechanisms of PostC are not completely elucidated, a series of mechanisms have been found to connect with PostC in the central nervous system, such as regulating synaptic signaling, attenuating oxidative stress and inflammation, maintaining mitochondrial integrity, inhibiting endoplasmic reticulum stress, regulating autophagy, activating PI3K/Akt pathway, inhibiting apoptosis, protecting neurovascular unit, etc. Based on these multiple protective mechanisms, PostC has high expectations to translate to the clinic, but a few issues should be resolved such as the time window, risks, efficiency, the impact of age, gender, hypertension, hyperlipidemia and t-PA, and clinical maneuverability. Even so, PostC could soon be at the bedside if the clinical trials are carefully planned.

A novel neuroprotective strategy for ischemic stroke: transient mild acidosis treatment by CO2 inhalation at reperfusion.

Acidosis is one of the key components in cerebral ischemic postconditioning that has emerged recently as an endogenous strategy for neuroprotection. We set out to test whether acidosis treatment at reperfusion can protect against cerebral ischemia/reperfusion injury. Adult male C57BL/6 J mice were subjected to 60-minute middle cerebral arterial occlusion followed by 24-hour reperfusion. Acidosis treatment by inhaling 10%, 20%, or 30% CO2 for 5 or 10 minutes at 5, 50, or 100 minutes after reperfusion was applied. Our results showed that inhaling 20% CO2 for 5 minutes at 5 minutes after reperfusion-induced optimal neuroprotection, as revealed by reduced infarct volume. Attenuating brain acidosis with NaHCO3 significantly compromised the acidosis or ischemic postconditioning-induced neuroprotection. Consistently, both acidosis-treated primary cultured cortical neurons and acute corticostriatal slices were more resistant to oxygen-glucose deprivation/reperfusion insult. In addition, acidosis inhibited ischemia/reperfusion-induced apoptosis, caspase-3 expression, cytochrome c release to cytoplasm, and mitochondrial permeability transition pore (mPTP) opening. The neuroprotection of acidosis was inhibited by the mPTP opener atractyloside both in vivo and in vitro. Taken together, these findings indicate that transient mild acidosis treatment at reperfusion protects against cerebral ischemia/reperfusion injury. This neuroprotection is likely achieved, at least partly, by inhibiting mPTP opening and mitochondria-dependent apoptosis.

Transient lack of glucose but not O2 is involved in ischemic postconditioning-induced neuroprotection.

AIM: Cerebral ischemic postconditioning has emerged recently as a kind of endogenous strategy for neuroprotection. We set out to test whether hypoxia or glucose deprivation (GD) would substitute for ischemia in postconditioning. METHODS: Adult male C57BL/6J mice were treated with postconditioning evoked by ischemia (bilateral common carotid arteries occlusion) or hypoxia (8% O(2) ) after 45-min middle cerebral arterial occlusion. Corticostriatal slices from mice were subjected to 1-min oxygen-glucose deprivation (OGD), GD, or oxygen deprivation (OD) postconditioning at 5 min after 15-min OGD. RESULTS: Hypoxic postconditioning did not decrease infarct volume or improve neurologic function at 24 h after reperfusion, while ischemic postconditioning did. Similarly, OGD and GD but not OD postconditioning attenuated the OGD/reperfusion-induced injury in corticostriatal slices. The effective duration of low-glucose (1 mmol/L) postconditioning was longer than that of OGD postconditioning. Moreover, OGD and GD but not OD postconditioning reversed the changes of glutamate, GABA, glutamate transporter-1 protein expression, and glutamine synthetase activity induced by OGD/reperfusion. CONCLUSIONS: These results suggest that the transient lack of glucose but not oxygen plays a key role in ischemic postconditioning-induced neuroprotection, at least partly by regulating glutamate metabolism. Low-glucose postconditioning might be a clinically safe and feasible therapeutic approach against cerebral ischemia/reperfusion injury.

Chronic h1-antihistamine treatment increases seizure susceptibility after withdrawal by impairing glutamine synthetase.

AIM: To investigate the effect of chronic H1-antihistamine treatment on seizure susceptibility after drug withdrawal in nonepileptic rats and to further study its relation to glutamine synthetase (GS), which is the key enzyme for glutamate metabolism and gamma aminobutyric acid (GABA) synthesis. METHODS: After drug withdrawal from a 2-week treatment with diphenhydramine or pyrilamine, seizure susceptibility was determined by amygdaloid kindling or pentylenetetrazol model; meanwhile, the GS expression or activity was analyzed. The glutamine, glutamate, and GABA contents were measured by high-performance liquid chromatography. RESULTS: Seizure susceptibility significantly increased in amygdaloid kindling and pentylenetetrazol model 10 days after drug withdrawal from a 2-week treatment with H1-antihistamines. Meanwhile, GS activity and expression in the cortex or hippocampus decreased simultaneously with a marked decline of glutamine and GABA content. Comparable inhibition of GS activity by methionine sulfoximine was also sufficient to increase the susceptibility, while supplementation with glutamine reversed the high susceptibility 10 days after diphenhydramine withdrawal. Moreover, the seizure susceptibility increased 10 days after diphenhydramine withdrawal in wild-type mice but not in histidine decarboxylase knockout mice, which lack histamine. CONCLUSIONS: Chronic H1-antihistamine treatment produces long-lasting increase in seizure susceptibility in nonepileptic rodents after drug withdrawal and its mechanism involves impairment of GS through blocking the action of histamine.

Acidic preconditioning protects against ischemia-induced brain injury.

Ischemic preconditioning protects against cerebral ischemia. Recent investigations indicated that acidic preconditioning (APC) protects against ischemia-induced cardiomyocytes injury. However, it is not clear whether APC can protect against cerebral ischemia. To address this issue, C57BL/6 mice were exposed 3 times at 10-min intervals to a normoxic atmosphere containing 20% CO(2) for 5 min before being further subjected to bilateral common carotid artery occlusion. APC reversed the ischemia-induced brain injury as revealed by improved performance in passive avoidance experiments and decreased neuron loss in the hippocampal CA1 region. Consistently, both APC-treated brain slices and primary cultured neurons were more resistant to oxygen-glucose-deprivation (OGD)-induced injury, in a pH- and time-dependent manner, as revealed by reversed cell/tissue viability. In addition, the APC treatment prevented OGD-induced mitochondrial transmembrane potential loss and apoptosis, which was inhibited by the mitochondrial permeability transport pore opener atractyloside. Taken together, these findings indicated that APC protects against ischemia-induced neuronal injury. The beneficial effects may be attributed, at least in part, to decreased mitochondria-dependent neuronal apoptosis.

Activation of the central histaminergic system is involved in hypoxia-induced stroke tolerance in adult mice.

We hypothesized that activation of the central histaminergic system is required for neuroprotection induced by hypoxic preconditioning. Wild-type (WT) and histidine decarboxylase knockout (HDC-KO) mice were preconditioned by 3 hours of hypoxia (8% O(2)) and, 48 hours later, subjected to 30 minutes of middle cerebral artery (MCA) occlusion, followed by 24 hours of reperfusion. Hypoxic preconditioning improved neurologic function and decreased infarct volume in WT or HDC-KO mice treated with histamine, but not in HDC-KO or WT mice treated with α-fluoromethylhistidine (α-FMH, an inhibitor of HDC). Laser-Doppler flowmetry analysis showed that hypoxic preconditioning ameliorated cerebral blood flow (CBF) in the periphery of the MCA territory during ischemia in WT mice but not in HDC-KO mice. Histamine decreased in the cortex of WT mice after 2, 3, and 4 hours of hypoxia, and HDC activity increased after 3 hours of hypoxia. Vascular endothelial growth factor (VEGF) mRNA and protein expressions showed a greater increase after hypoxia than those in HDC-KO or α-FMH-treated WT mice. In addition, the VEGF receptor-2 antagonist SU1498 prevented the protective effect of hypoxic preconditioning in infarct volume and reversed increased peripheral CBF in WT mice. Therefore, endogenous histamine is an essential mediator of hypoxic preconditioning. It may function by enhancing hypoxia-induced VEGF expression.

Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity.

Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of Delta Psi(m) and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen-glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.

[Effect of endogenous histamine on ischemic preconditioning induced cerebral ischemic tolerance].

OBJECTIVE: To investigate the effect of endogenous histamine on ischemic preconditioning induced cerebral ischemic tolerance in rats. METHODS: Wild-type (WT) mice and histidine decarboxylase knock-out (HDC-KO) mice were preconditioned by bilateral carotid artery occlusion (BCCAO) for 6, 10,or 14 min and reperfused for 48 h,then subjected to permanent BCCAO and the survival time of WT and HDC-KO mice subjected to permanent BCCAO was observed. Histamine levels in the hypothalamus, hippocampus, striatum and cortex at 0.5 h,5 h or 48 h after 10 min BCCAO were determined with high-performance liquid chromatography. RESULT: Ten minutes ischemic preconditioning significantly prolonged the survival time of WT mice subjected to permanent BCCAO. However,in HDC-KO mice, the ischemic tolerance was not induced with 10 min preconditioning. The histamine levels at 0.5 h or 48 h increased after 10 min preconditioning, but not at 5 h. CONCLUSION: Endogenous histamine in brain may be an essential mediator in ischemic preconditioning induced cerebral ischemic tolerance.