Neurons and Neuronal Stem Cells Survive in Glucose-Free Lactate and in High Glucose Cell Culture Medium During Normoxia and Anoxia

Several questions concerning the survival of isolated neurons and neuronal stem and progenitor cells (NPCs) have not been answered in the past: (1) If lactate is discussed as a major physiological substrate of neurons, do neurons and NPCs survive in a glucose-free lactate environment? (2) If elevated levels of glucose are detrimental to neuronal survival during ischemia, do high concentrations of glucose (up to 40 mmol/L) damage neurons and NPCs? (3) Which is the detrimental factor in oxygen glucose deprivation (OGD), lack of oxygen, lack of glucose, or the combination of both? Therefore, in the present study, we exposed rat cortical neurons and NPCs to different concentrations of d-glucose ranging from 0 to 40 mmol/L, or 10 and 20 mmol/L l-lactate under normoxic and anoxic conditions, as well as in OGD. After 24 h, we measured cellular viability by biochemical assays and automated cytochemical morphometry, pH values, bicarbonate, lactate and glucose concentrations in the cell culture media, and caspases activities. We found that (1) neurons and NPCs survived in a glucose-free lactate environment at least up to 24 h, (2) high glucose concentrations >5 mmol/L had no effect on cell viability, and (3) cell viability was reduced in normoxic glucose deprivation to 50% compared to 10 mmol/L glucose, whereas cell viability in OGD did not differ from that in anoxia with lactate which reduced cell viability to 30%. Total caspases activities were increased in the anoxic glucose groups only. Our data indicate that (1) neurons and NPCs can survive with lactate as exclusive metabolic substrate, (2) the viability of isolated neurons and NPCs is not impaired by high glucose concentrations during normoxia or anoxia, and (3) in OGD, low glucose concentrations, but not low oxygen levels are detrimental for neurons and NPCs.     

白藜芦醇对氧糖剥夺／再灌注损伤的PCI2细胞的保护作用及其作用机制

目的：探讨白藜芦醇对氧糖剥夺／再灌注（OGD／R）损伤的PCI2细胞的保护作用及其机制。方法：体外培养PCI2细胞,分为对照组,白藜芦醇组,OGD／R组及OGD／R＋白藜芦醇组。以改良的噻唑蓝法测定细胞活性,采用AnnexinV—FITC／PI双染法检测细胞的凋亡率,用双氯罗丹明（DHR）检测细胞内活性氧簇（Ros）的水平,采用蛋白印迹法（westemblot）分析SIRTl的蛋白表达情况。结果：与对照组相比,经过OGD／R损伤后,细胞活力显著降低。而在OGD／R的同时给予10μmol／L的白藜芦醇处理。可以明显提高细胞活力。流式细胞仪检测发现,10μmol／L的白藜芦醇可以显著地减少OGD／R引起的细胞凋亡,抑制细胞内的ROS产生。westemblot的结果提示,与对照组比较,白藜芦醇可提高SIRTl的蛋白表达水平。结论：白藜芦醇可以通过抑制ROS的产生和上调SIRTl的表达等机制而发挥其对抗氧糖剥夺／再灌注损伤的神经保护性作用。     

Time-Dependent Changes in Apoptosis Upon Autophagy Inhibition in Astrocytes Exposed to Oxygen and Glucose Deprivation

Recent studies have implicated the role of autophagy in brain ischemia pathophysiology. However, it remains unclear whether autophagy activation is protective or detrimental to astrocytes undergoing ischemic stress. This study evaluated the influence of ischemia-induced autophagy on cell death and the course of intrinsic and extrinsic apoptosis in primary cultures of rat cortical astrocytes exposed to combined oxygen-glucose deprivation (OGD). The role of autophagy was assessed by pharmacological inhibition with 3-methyladenine (3-MA). Cell viability was evaluated by measuring LDH release and through the use of the alamarBlue Assay. Apoptosis and necrosis were determined by fluorescence microscopy after Hoechst 33,342 and propidium iodide staining, respectively. The levels of apoptosis-related proteins were analyzed by immunoblotting. The downregulation of autophagy during OGD resulted in decreased cell viability and time-dependent changes in levels of apoptosis and necrosis. After short-term OGD (1, 4 h), cells treated with 3-MA showed higher level of cleaved caspase 3 compared with control cells. This result was consistent with an evaluation of apoptotic cell number by fluorescence microscopy. However, after prolonged exposure to OGD (8, 24 h), the number of apoptotic astrocytes (microscopically evaluated) did not differ or was even lower (as marked by caspase 3) in the presence of the autophagy inhibitor in comparison to the control. A higher level of necrosis was observed in 3-MA-treated cells compared to non-treated cells after 24 h OGD. The downregulation of autophagy caused time-dependent changes in both extrinsic (cleaved caspase 8, TNFα) and intrinsic (cleaved caspase 9) apoptotic pathways. Our results strongly indicate that the activation of autophagy in astrocytes undergoing ischemic stress is an adaptive mechanism, which allows for longer cell survival by delaying the initiation of apoptosis and necrosis.     

Beneficial Effect of Astragalosides on Stroke Condition Using PC12 Cells under Oxygen Glucose Deprivation and Reperfusion

Astragalosides (AST) are reported to be neuroprotective in focal cerebral ischemic models in vivo. In this study, the direct effect of AST against oxygen and glucose deprivation (OGD) including neuronal injury and the underlying mechanisms in vitro were investigated. 5 h OGD followed by 24 h of reperfusion [adding back oxygen and glucose (OGD-R)] was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. AST (1, 100, and 200 µg/mL) were added to the culture after 5 h of the OGD ischemic insult and was present during the reoxygenation phases. A key finding was that OGD-R decreased cell viability, increased lactate dehydrogenase, increased reactive oxygen species, apoptosis, autophagy, functional impairment of mitochondria, and endoplasmic reticulum stress in PC12 cells, all of which AST treatment significantly reduced. In addition, AST attenuated OGD-R-induced cell loss through P38 MAPK activation a neuroprotective effect blunted by SB203580, a specific inhibitor of P38 MAPK. Our data suggest that both apoptosis and autophagy are important characteristics of OGD-R-induced PC12 death and that treating PC12 cells with AST blocked OGD-R-induced apoptosis and autophagy by suppressing intracellular oxidative stress, functional impairment of mitochondria, and endoplasmic reticulum stress. Our data provide identification of AST that can concomitantly inhibit multiple cells death pathways following OGD injuries in neural cells.     

Injury to primary cultures of rat heart endothelial cells by hypoxia and glucose deprivation

Primary cultures of rat heart endothelial cells were subjected to simulated conditions of ischemia: hyposia and glucose deprivation for 4 and 24 hr. Cellular injury was evaluated by measuring changes in viability, total protein, cellular morphology, and leakage of cytoplasmic enzymes from the cells into the culture medium. Deprivation of oxygen and glucose for 4 or 24 hr did not lethally injure the cells as noted by no change in cell viability, morphology, and total protein when compared to controls. However, reversible or non-lethal cellular injury was produced as reflected by a significant release of lactate dehydrogenase (LDH) from the cells into the medium after treatment with hypoxia and glucose deprivation for 4 or 24 hr. When the cultures were deprived of glucose, but were oxygenated, cellular injury was not evident after 24 hr. Deprivation of oxygen but not glucose resulted in significant loss of LDH after 4 or 24 hr. When the cultures were allowed to recover after oxygen and glucose deprivation in complete medium containing 1000 mg glucose per 1 and a normal atmosphere of 20% O2, they had levels of LDH leakage comparable to those of control cultures.     

Validation of organotypical hippocampal slice cultures as an ex vivo model of brain ischemia: different roles of NMDA receptors in cell death signalling after exposure to NMDA or oxygen and glucose deprivation

N-Methyl-D-aspartate receptors (NMDARs) are essential mediators of synaptic plasticity under normal physiological conditions. During brain ischemia, these receptors are excessively activated due to glutamate overflow and mediate excitotoxic cell death. Although organotypical hippocampal slice cultures are widely used to study brain ischemia in vitro by induction of oxygen and glucose deprivation (OGD), there is scant data regarding expression and functionality of NMDARs in such slice cultures. Here, we have evaluated the contribution of NMDARs in mediating excitotoxic cell death after exposure to NMDA or OGD in organotypical hippocampal slice cultures after 14 days in vitro (DIV14). We found that all NMDAR subunits were expressed at DIV14. The NMDARs were functional and contributed to cell death, as evidenced by use of the NMDAR antagonist MK-801 (dizocilpine). Excitotoxic cell death induced by NMDA could be fully antagonized by 10 μM MK-801, a dose that offered only partial protection against OGD-induced cell death. Very high concentrations of MK-801 (50–100 μM) were required to counteract cell death at long delays (48–72 h) after OGD. The relative high dose of MK-801 needed for long-term protection after OGD could not be attributed to down-regulation of NMDARs at the gene expression level. Our data indicate that NMDAR signaling is just one of several mechanisms underlying ischemic cell death and that prospective cytoprotective therapies must be directed to multiple targets.     

Intermittent Hypothermia Is Neuroprotective in an in vitro Model of Ischemic Stroke

Objective: To investigate whether the intermittent hypothermia (IH) protects neurons against ischemic insult and the potential molecular targets using an in vitro ischemic model of oxygen glucose deprivation (OGD).Methods: Fetal rat cortical neurons isolated from Day E18 rat embryos were subjected to 90-min OGD and hypothermia treatments during reoxygenation before examining the changes in microscopic morphology, cell viability, microtubule- associated protein 2 (MAP-2) release, intracellular pH value and calcium, reactive oxygen species (ROS) generation, mitochondrial membrane potential (△Ψm) and neuronal death using cell counting kit (CCK-8), enzyme-linked immunosorbent assay (ELISA), BCECF AM, Fluo-3 AM, DCFH-DA and dihydroethidium (DHE), JC-1 staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively.Results: 90-min OGD induced morphologic abnormalities, cell viability decline, MAP-2 release, intracellular acidosis, calcium overload, increased ROS generation, △Ψm decrease and cell death in primary neurons, which was partially inhibited by continuous hypothermia (CH) and intermittent hypothermia (IH). Interestingly, 6-h CH was insufficient to reduce intracellular calcium overload and stabilize mitochondrial membrane potential (△Ψm), while 12-h CH was effective in reversing the above changes. All IH treatments (6×1 h, 4×1.5 h or 3×2 h) effectively attenuated intracellular free calcium overload, inhibited ROS production, stabilized mitochondrial membrane potential (△Ψm) and reduced delayed cell death in OGD-treated cells. However, only IH intervals longer than 1.5 h appeared to be effective in preventing cell viability loss and intracellular pH decline.Conclusion: Both CH and IH were neuroprotective in an in vitro model of ischemic stroke, and in spite of shorter hypothermia duration, IH could provide a comparable neuroprotection to CH.     

Neuropeptide Y-Y1 receptor agonist worsens while antagonist improves survival of cultured Y1-expressing neuronal cells following oxygen and glucose deprivation

In this in vitro study, we investigated the influence of neuropeptide Y (NPY) Y1 receptor activation or inhibition on the viability of cultured neuronal or glial cells following oxygen glucose deprivation (OGD). Viability of cultured cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. When compared to the vehicle-treated control group, treatment with NPY or [Leu³¹,Pro³⁴]-NPY (Y1 agonist) reduced viability of cultured SK-N-MC (Y1-expressing) human neuronal cells at 24 h after 1 h of OGD, while BIBP3226 (Y1 antagonist) improved viability. Except at the highest concentration of NPY used in the study, treatment with NPY or NPY3-36 (Y2 agonist) did not influence viability of cultured SH-SY5Y (Y2-expressing) human neuronal cells at 24 h after 1 h of OGD. In addition, treatment with NPY, [Leu³¹,Pro³⁴]-NPY, NPY3-36, or BIBP3226 did not affect viability of cultured primary astrocytes at 24 h after 4 h of OGD. The present results agree with those of a recent in vivo study. Activation of NPY-Y1 receptors may mediate ischemic pathophysiological processes, and inhibiting the Y1 receptors may be protective. The combination of OGD and cultured neuronal cells may be useful in future studies on the neuroprotective and harmful mechanisms of NPY-Y1 receptor inhibition and activation during ischemia, respectively.     

Morphological and functional changes in rat hippocampal slice cultures after short-term oxygen-glucose deprivation

To study effects of short-term cerebral ischemia, hippocampal slice cultures were subjected to oxygen and glucose deprivation (OGD) followed by a period of normoxic reoxygenation. Propidium iodide staining, and MTT/formazan-assay were used to evaluate cell viability and metabolic activity. CA1 pyramidal cells were analyzed at the light- and electron microscopic levels. Cell damage was found to be insignificant during the first hour after 10 min OGD but profound following 4 h, showing delayed neuronal cell damage caused by short-term OGD. Our model can be used to characterize the mechanisms of cell damage caused by mild cerebral ischemia. These data might apply to further development of neuroprotective tools for the treatment of brain diseases.     

Pyrroloquinoline quinone inhibits oxygen/glucose deprivation-induced apoptosis by activating the PI3K/AKT pathway in cardiomyocytes

The purposes of this study were to examine the protective effect of pyrroloquinoline quinone (PQQ) on oxygen/glucose deprivation (OGD)-induced injury to H9C2 rat cardiomyocytes and to investigate the mechanism. Using H9C2 cells cultured in vitro, we examined changes in cell viability with an MTT assay at 12, 24, and 48 h after injury induced by OGD. Various concentrations of PQQ (1, 10, and 100 μM) were added, and the effect of PQQ on cell viability after OGD was assessed using the MTT assay. Thus, the optimal concentration of PQQ for the protection of cardiomyocytes against oxygen and glucose deprivation injury was determined. We also used flow cytometry analysis to examine the effect of PQQ on H9C2 cells with OGD-induced injury. The molecular probe 2′,7′-dichlorofluorescin diacetate was used to label the H9C2 cells, and flow cytometry was used to detect the effect of PQQ on reactive oxygen species (ROS) content. After labeling the H9C2 cells using a mitochondrial green fluorescent probe (Mito-Tracker Green), we measured the change in the mitochondrial content of PQQ-treated H9C2 cells. Western blotting was used to examine the effect of PQQ on the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in the H9C2 cells. The results of the MTT assay showed that 48 h of OGD significantly injured the H9C2 cells (p < 0.01) and that treatment with 100 μM PQQ effectively decreased the level of OGD-induced injury (p < 0.01). The results of the flow cytometry analysis showed that PQQ significantly reduced apoptosis in H9C2 cells subjected to OGD (p < 0.05). In addition, OGD significantly increased the ROS level in H9C2 cells (p < 0.01), and PQQ significantly inhibited this increase (p < 0.05). The results of the Mito-Tracker Green staining suggested that PQQ effectively inhibited the decrease in mitochondrial content caused by OGD (p < 0.05). Western blot analysis showed that PQQ partially reversed the decrease in Akt phosphorylation that was caused by OGD (p < 0.05). PQQ treatment dose-dependently protects H9C2 cells from OGD-induced injury by reducing apoptosis, decreasing intracellular ROS levels, and rescuing the OGD-induced decrease in mitochondrial content. The protective effect of PQQ may be related to its effects on the PI3K/Akt pathway.     

Hydroxysafflor Yellow A Protects PC12 Cells Against the Apoptosis Induced by Oxygen and Glucose Deprivation

Hydroxysafflor yellow A (HSYA) was reported neuroprotective under several ischemic models in vivo. In this study, the direct effect of HSYA against oxygen–glucose deprivation (OGD) inducing acute neuronal injury and the underling mechanisms in vitro were investigated. Four-hour oxygen and glucose deprivation (OGD) followed by 20 h reperfusion (adding back oxygen and glucose, OGD-R) was used to induce in vitro ischemia reperfusion injury in differentiated rat pheochromocytoma PC12 cells. HSYA (1, 10, and 100 μmol/l) was added to the cultures 30 min prior to the ischemic insult and was present during OGD and reoxygenation phases. The survival rate of PC12 cells was detected by MTT assay. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) activity were elevated by biochemical method. Hoechst 33258 staining and flow cytometric analysis were used to detect apoptosis; western blotting was used to detect the expression of Bcl-2, Bax, and Cytochrome C protein. The activity of caspase-3 was assessed by colorimetry. HSYA concentration-dependently attenuated neuronal damage with characteristics of increasing injured neuronal absorbance of MTT, decreasing cell apoptosis, and antagonizing decreases in SOD activity and increase in MDA level induced by OGD-R. Moreover, the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol and the consequent activation of caspase-3 were reversed by HSYA in a dose-dependent manner. These results suggest that apoptosis is an important characteristic of OGD-R-induced PC 12 death and that treatment of PC12 cells with HSYA can block OGD-R-induced apoptosis through suppression of intracellular oxidative stress and mitochondria dependent caspase cascade.     

Oxygen glucose deprivation causes mitochondrial dysfunction in cultivated rat hippocampal slices: Protective effects of CsA,its immunosuppressive congener [D-Ser]8CsA,the novel non-immunosuppressive cyclosporin derivative Cs9, and the NMDA receptor antagonist MK 801

We have introduced a sensitive method for studying oxygen/glucose deprivation (OGD)-induced mitochondrial alterations in homogenates of organotypic hippocampal slice cultures (slices) by high-resolution respirometry. Using this approach, we tested the neuroprotective potential of the novel non-immunosuppressive cyclosporin (CsA) derivative Cs9 in comparison with CsA, the immunosuppressive CsA analog [D-Ser]⁸CsA, and MK 801, a N-methyl-d-aspartate (NMDA) receptor antagonist. OGD/reperfusion reduced the glutamate/malate dependent (and protein-related) state 3 respiration to 30% of its value under control conditions. All of the above drugs reversed this effect, with an increase to > 88% of the value for control slices not exposed to OGD. We conclude that Cs9, [D-Ser]⁸CsA, and MK 801, despite their different modes of action, protect mitochondria from OGD-induced damage.     

Endothelial cells regulate p53-dependent apoptosis of neural progenitors after irradiation

Endothelial cells represent an important component of the neurogenic niche and may regulate self-renewal and differentiation of neural progenitor cells (NPCs). Whether they have a role in determining the apoptotic fate of NPCs after stress or injury is unclear. NPCs are known to undergo p53-dependent apoptosis after ionizing radiation, whereas endothelial cell apoptosis after irradiation is dependent on membrane acid sphingomyelinase (ASMase) and is abrogated in sphingomyelin phosphodiesterase 1 (smpd1)- (gene that encodes ASMase) deficient mice. Here we found that p53-dependent apoptosis of NPCs in vivo after irradiation was inhibited in smpd1-deficient mice. NPCs cultured from mice, wild type (+/+) or knockout (−/−), of the smpd1 gene, however, demonstrated no difference in apoptosis radiosensitivity. NPCs transplanted into the hippocampus of smpd1−/− mice were protected against apoptosis after irradiation compared with those transplanted into smpd1+/+ mice. Intravenous administration of basic fibroblast growth factor, which does not cross the blood–brain barrier, known to protect endothelial cells against apoptosis after irradiation also attenuated the apoptotic response of NPCs. These findings provide evidence that endothelial cells may regulate p53-dependent apoptosis of NPCs after genotoxic stress and add support to an important role of endothelial cells in regulating apoptosis of NPCs after injury or in disease.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) Signaling and Cell Death in the Immature Central Nervous System after Hypoxia-Ischemia and Inflammation

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family. The interaction of TRAIL with death receptor 4 (DR4) and DR5 can trigger apoptotic cell death. The aim of this study was to investigate the role of TRAIL signaling in neonatal hypoxia-ischemia (HI). Using a neonatal mouse model of HI, mRNA, and protein expression of TRAIL, DR5 and the TRAIL decoy receptors osteoprotegerin (OPG), mDcTRAILR1, and mDcTRAILR2 were determined. In vitro, mRNA expression of these genes was measured in primary neurons and oligodendrocyte progenitor cells (OPCs) after inflammatory cytokine (TNF-α/IFN-γ) treatment and/or oxygen and glucose deprivation (OGD). The toxicity of these various paradigms was also measured. The expression of TRAIL, DR5, OPG, and mDcTRAILR2 was significantly increased after HI. In vitro, inflammatory cytokines and OGD treatment significantly induced mRNAs for TRAIL, DR5, OPG, and mDcTRAILR2 in primary neurons and of TRAIL and OPG in OPCs. TRAIL protein was expressed primarily in microglia and astroglia, whereas DR5 co-localized with neurons and OPCs in vivo. OGD enhanced TNF-α/IFN-γ toxicity in both neuronal and OPC cultures. Recombinant TRAIL exerted toxicity alone or in combination with OGD and TNF-α/IFN-γ in primary neurons but not in OPC cultures. The marked increases in the expression of TRAIL and its receptors after cytokine exposure and OGD in primary neurons and OPCs were similar to those found in our animal model of neonatal HI. The toxicity of TRAIL in primary neurons suggests that TRAIL signaling participates in neonatal brain injury after inflammation and HI.     

Oxygen and glucose deprivation induces mitochondrial dysfunction and oxidative stress in neurones but not in astrocytes in primary culture

In order to investigate the potential neuroprotective role played by glucose metabolism during brain oxygen deprivation, the susceptibility of cultured neurones and astrocytes to 1 h of oxygen deprivation (hypoxia) or oxygen and glucose deprivation (OGD) was examined. OGD, but not hypoxia, promotes dihydrorhodamine 123 and glutathione oxidation in neurones but not in astrocytes reflecting free radical generation in the former cells. A specific loss of mitochondrial complex-I activity, mitochondrial membrane potential collapse, ATP depletion and necrosis occurred in the OGD neurones, but not in the OGD astrocytes. Furthermore, superoxide anion but not nitric oxide formation was responsible for these effects. OGD decreased neuronal but not astrocytic NADPH concentrations; this was not observed in hypoxia and was independent of superoxide or nitric oxide formation. These results suggest that glucose metabolism would supply NADPH, through the pentose-phosphate pathway, aimed at preventing oxidative stress, mitochondrial damage and neurotoxicity during oxygen deprivation to neural cells.     

Mesodermal cell types induce neurogenesis from adult human hippocampal progenitor cells

Neurogenesis in the adult human brain occurs within two principle neurogenic regions, the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Recent reports demonstrated the isolation of human neuroprogenitor cells (NPCs) from these regions, but due to limited tissue availability the knowledge of their phenotype and differentiation behavior is restricted. Here we characterize the phenotype and differentiation capacity of human adult hippocampal NPCs (hNPCs), derived from patients who underwent epilepsy surgery, on various feeder cells including fetal mixed cortical cultures, mouse embryonic fibroblasts (MEFs) and PA6 stromal cells. Isolated hNPCs were cultured in clonal density by transferring the cells to serum-free media supplemented with FGF-2 and EGF in 3% atmospheric oxygen. These hNPCs showed neurosphere formation, expressed high levels of early neuroectodermal markers, such as the proneural genes NeuroD1 and Olig2, the NSC markers Nestin and Musashi1, the proliferation marker Ki67 and significant activity of telomerase. The phenotype was CD15low/-, CD34-, CD45- and CD133-. After removal of mitogens and plating them on poly D-lysine, they spontaneously differentiated into a neuronal (MAP2ab+), astroglial (GFAP+), and oligodendroglial (GalC+) phenotype. Differentiated hNPCs showed functional properties of neurons, such as sodium channels, action potentials and production of the neurotransmitters glutamate and GABA. Co-culture of hNPCs with fetal cortical cultures, MEFs and PA6 cells increased neurogenesis of hNPCs in vitro, while only MEFs and PA6 cells also led to a morphological and functional neurogenic maturation. Together we provide a first detailed characterization of the phenotype and differentiation potential of human adult hNPCs in vitro. Our findings reinforce the emerging view that the differentiation capacity of adult hNPCs is critically influenced by non-neuronal mesodermal feeder cells.     

Progesterone Attenuates Aquaporin-4 Expression in an Astrocyte Model of Ischemia/Reperfusion

Previous studies have suggested that progesterone may be involved in neuroprotection by preventing brain edema. In this study, we assessed the effects of progesterone on aquaporin-4 (AQP4) expression in an ischemia/reperfusion model of cultured rat astrocytes, and further explored the possible role of the protein kinase C (PKC) pathway in this course. We evaluate primary culture astrocytes exposed to 4 h oxygen–glucose deprivation (OGD) followed by 24 h reperfusion (OGD4h/R24h) as a means of simulating cortex ischemia and reperfusion, and test the effect of progesterone on AQP4 expression in response to OGD4h/R24h. Besides, the cell viability was assessed by MTT reduction and lactate dehydrogenase release assay, accompanied by cell morphology survey. At a concentration of 1 and 2 μM, progesterone significantly attenuated AQP4 at the level of both protein and mRNA and ameliorated the cell viability of astrocytes from OGD/reperfusion injury. Moreover, this effect was blocked by the PKC inhibitor Ro31-8220, which was employed before the OGD. These results indicate that progesterone exerts the protective effects and attenuates AQP4 expression in an astrocyte model of ischemia/reperfusion depending on the PKC signal pathway.     

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model

Purpose

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure “transplant conditions” (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

Methods

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

Results

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

Conclusion

Pre-confluent cells should be used to maximize graft cell viability.     

Cryopreservation at −20 and −70 °C of Pleurotus ostreatus on Grains

Alternative substrates for cryopreservation at −20 °C have been little explored for basidiomycetes and could bring new possibilities of lower cost cryopreservation. Nevertheless, freezing temperatures between −15 and −60 °C are very challenging because they frequently result in cryoinjuries. The objective of this study was to evaluate substrates associated to cryoprotective agents for Pleurotus ostreatus cryopreservation at −20 or −70 °C in order to develop alternative techniques for basidiomycete cryopreservation. P. ostreatus was grown on potato dextrose agar or whole grains of oat, wheat, rice or millet and transferred to cryovials with cryoprotective solution with 1 % dimethyl sulfoxide, 5 % glycerol, 10 % saccharose, 4 % glucose, 6 % polyethylene glycol-6000 or 5 % malt extract. The mycelium in the cryovials were cryopreserved at −20 or −70 °C and recovered for evaluation of the mycelial growth viability after 1 and 3 years. Both substrates and cryoprotectants affect the viability of the mycelial growth cryopreserved at −20 or −70 °C; wheat grains combined with cryoprotectants such as saccharose or glucose are effective for keeping mycelium viable after cryopreservation at −20 °C for 1 or 3 years; for cryopreservation at −70 °C after 1 or 3 years, any substrate combined with any cryoprotectant is effective for preserving the mycelium viable, except for millet grains with polyethylene glycol after 3 years; semi-permeable cryoprotective agents such as saccharose and glucose are the most effective for cryopreservation at −20 or −70 °C for at least 3 years.     

Ventral tegmental area/substantia nigra and prefrontal cortex rodent organotypic brain slices as an integrated model to study the cellular changes induced by oxygen/glucose deprivation and reperfusion: Effect of neuroprotective agents

Unveiling the roles of distinct cell types in brain response to insults is a partially unsolved challenge and a key issue for new neuroreparative approaches. In vivo models are not able to dissect the contribution of residential microglia and infiltrating blood-borne monocytes/macrophages, which are fundamentally undistinguishable; conversely, cultured cells lack original tissue anatomical and functional complexity, which profoundly alters reactivity. Here, we tested whether rodent organotypic co-cultures from mesencephalic ventral tegmental area/substantia nigra and prefrontal cortex (VTA/SN-PFC) represent a suitable model to study changes induced by oxygen/glucose deprivation and reperfusion (OGD/R). OGD/R induced cytotoxicity to both VTA/SN and PFC slices, with higher VTA/SN susceptibility. Neurons were highly affected, with astrocytes and oligodendrocytes undergoing very mild damage. Marked reactive astrogliosis was also evident. Notably, OGD/R triggered the activation of CD68-expressing microglia and increased expression of Ym1 and Arg1, two markers of “alternatively” activated beneficial microglia. Treatment with two well-known neuroprotective drugs, the anticonvulsant agent valproic acid and the purinergic P2-antagonist PPADS, prevented neuronal damage. Thus, VTA/SN-PFC cultures are an integrated model to investigate OGD/R-induced effects on distinct cells and easily screen neuroprotective agents. The model is particularly adequate to dissect the microglia phenotypic shift in the lack of a functional vascular compartment.