Arctic ground squirrel neuronal progenitor cells resist oxygen and glucose deprivation-induced death

AIM: To investigate the influence of ischemia/reperfusion on arctic ground squirrel(AGS) neuronal progenitor cells(NPCs), we subjected these cultured cells to oxygen and glucose deprivation.METHODS: AGS NPCs were expanded and differentiated into NPCs and as an ischemia vulnerable control, commercially available human NPCs(hNPCs) were seeded from thawed NPCs. NPCs, identified by expression of TUJ1 were seen at 14-21 d in vitro(DIV). Cultures were exposed to control conditions, hypoxia, oxygen and glucose deprivation or glucose deprivation alone or following return to normal conditions to model reperfusion. Cell viability and death were assessed from loss of ATP as well as from measures of alamarB lue~ and lactate dehydrogenase in the media and from counts of TUJ1 positive cells using immunocytochemistry. Dividing cells were identified by expression of Ki67 and phenotyped by double labeling with GFAP, MAP2 ab or TUJ1. RESULTS: We report that when cultured in NeuraLife~(TM), AGS cells remain viable out to 21 DIV, continue to express TUJ1 and begin to express MAP2 ab. Viability of hN PCs assessed by fluorescence alamarB lue(arbitrary units) depends on both glucose and oxygen availability [viability of hNPCs after 24 h oxygen glucose deprivation(OGD) with return of oxygen and glucose decreased from 48151 ± 4551 in control cultures to 43481 ± 2413 after OGD, P 0.05]. By contrast, when AGS NPCs are exposed to the same OGD with reperfusion at 14 DIV, cell viability assessed by alamar Blue increased from 165305 ± 11719 in control cultures to 196054 ± 13977 after OGD. Likewise AGS NPCs recovered ATP(92766 ± 6089 in control and 92907 ± 4290 after modeled reperfusion; arbitrary luminescence units), and doubled in the ratio of TUJ1 expressing neurons to total dividing cells(0.11 ± 0.04 in control cultures vs 0.22 ± 0.2 after modeled reperfusion, P 0.05). Maintaining AGS NPCs for a longer time in culture lowered resistance to injury, however, did not impair proliferation of NPCs relative to other cell lineages after oxygen deprivation followed by re-oxygenation.CONCLUSION: Ischemic-like insults decrease viability and increase cell death in cultures of human NPCs. Similar conditions have less affect on cell death and promote proliferation in AGS NPCs.     

TRPM4 activation by chemically- and oxygen deprivation-induced ischemia and reperfusion triggers neuronal death

Cerebral ischemia-reperfusion injury triggers a deleterious process ending in neuronal death. This process has two components, a glutamate-dependent and a glutamate-independent mechanism. In the glutamate-independent mechanism, neurons undergo a slow depolarization eventually leading to neuronal death. However, little is known about the molecules that take part in this process. Here we show by using mice cortical neurons in culture and ischemia-reperfusion protocols that TRPM4 is fundamental for the glutamate-independent neuronal damage. Thus, by blocking excitotoxicity, we reveal a slow activating, glibenclamide- and 9-phenanthrol-sensitive current, which is activated within 5 min upon ischemia-reperfusion onset. TRPM4 shRNA-based silenced neurons show a reduced ischemia-reperfusion induced current and depolarization. Neurons were protected from neuronal death up to 3 hours after the ischemia-reperfusion challenge. The activation of TRPM4 during ischemia-reperfusion injury involves the increase in both, intracellular calcium and H₂O₂, which may act together to produce a sustained activation of the channel.     

Alcohol absorption inhibitors from bay leaf (Laurus nobilis): structure-requirements of sesquiterpenes for the activity

Through a bioassay-guided separation using inhibitory activity on blood ethanol elevation in oral ethanol-loaded rat, various sesquiterpenes having an alpha-methylene-gamma-butyrolactone moiety, costunolide (1), dehydrocostus lactone (2), zaluzanin D (3), reynosin (4), santamarine (5), 3alpha-acetoxyeudesma-1,4(15),11(13)-trien-12,6alpha-+ ++olide (6) and 3-oxoeudesma-1,4,11(13)-trien-12,6alpha-olide (7), were isolated as the active principle from the leaves of Laurus nobilis (bay leaf, laurel). In order to characterize the structure requirement for the activity, several reduction products (2a-2d) and amino acid adducts (2e, 2f) of the alpha-methylene-gamma-butyrolactone moiety were synthesized from 2 and the inhibitory activities of these sesquiterpenes, together with alpha-methylene-gamma-butyrolactone (12) and its related compounds (13-16), were examined. These results indicated that the gamma-butyrolactone or gamma-butyrolactol moiety having alpha-methylene or alpha-methyl group was essential for the inhibitory activity on ethanol absorption. Since 1, 2 and 12 showed no significant effect on glucose absorption, these sesquiterpenes appeared to selectively inhibit ethanol absorption. In addition, the acute toxicities of 1 and 2 in a single oral administration were found to be lower than that of 12.     

Ultrastructure and development of oil cells in Laurus nobilis L. leaves

The oil cell development in Laurus nobilis leaves has been studied. At the early developmental stage, when the cell wall consists of the outer cellulose wall only, the oil cells differ from the neighbouring mesophyll cells in their larger size, lower starch content and in their plastid organization. After the deposition of the lamellated suberin layer and the inner cellulose layer, a wall protuberance (cupule) is formed on the periclinal wall facing the epidermis. From its reaction with periodic acid-hexamine-silver nitrate, it is suggested that the cupule is cellulosic. The portion of the inner cellulose wall layer bearing the cupule seems to contain patches of suberin. Plasmodesmata occur in special wall protuberances and appear to become occluded with age. The oil produced inside the protoplast is secreted to the outside of the plasmalemma, and accumulates as a drop at the place predetermined by the cupule. Except at the cupule, the oil drop is surrounded by the plasmalemma.     

First Report of Calonectria ilicicola Causing a New Disease on Laurus (Laurus nobilis) in Europe

Crown and root rot has been detected on potted Laurus nobilis plants in a nursery located in the Catania province (Italy). Perithecia referable to a Calonectria species were consistently detected on crowns and stems of symptomatic plants. Based on morphology, cultural features and molecular analysis, the species was identified as Calonectria ilicicola. Koch’s postulates were fulfilled by pathogenicity tests carried out on potted Laurus nobilis seedlings. To our knowledge, this is the first report of the occurrence of a disease caused by Ca. ilicicola on Laurus nobilis.     

Superoxide dismutase/catalase mimetics but not MAP kinase inhibitors are neuroprotective against oxygen/glucose deprivation-induced neuronal death in hippocampus

Although oxygen/glucose deprivation (OGD) has been widely used as a model of ischemic brain damage, the mechanisms underlying acute neuronal death in this model are not yet well understood. We used OGD in acute hippocampal slices to investigate the roles of reactive oxygen species and of the mitogen-activated protein kinases (MAPKs) in neuronal death. In particular, we tested the neuroprotective effects of two synthetic superoxide dismutase/catalase mimetics, EUK-189 and EUK-207. Acute hippocampal slices prepared from 2-month-old or postnatal day 10 rats were exposed to oxygen and glucose deprivation for 2 h followed by 2.5 h reoxygenation. Lactate dehydrogenase (LDH) release in the medium and propidium iodide (PI) uptake were used to evaluate cell viability. EUK-189 or EUK-207 applied during the OGD and reoxygenation periods decreased LDH release and PI uptake in slices from 2-month-old rats. EUK-189 or EUK-207 also partly blocked OGD-induced ATP depletion and extracellular signal-regulated kinases 1 and 2 (ERK1/2) dephosphorylation, and completely eliminated reactive oxygen species generation. The MEK inhibitor U0126 applied together with EUK-189 or EUK-207 completely blocked ERK1/2 activation, but had no effect on their protective effects against OGD-induced LDH release. U0126 alone had no effect on OGD-induced LDH release. EUK-207 had no effect on OGD-induced p38 or c-Jun N-terminal kinase dephosphorylation, and when the p38 inhibitor SB203580 was applied together with EUK-207, it had no effect on the protective effects of EUK-207. SB203580 alone had no effect on OGD-induced LDH release either. In slices from p10 rats, OGD also induced high-LDH release that was partly reversed by EUK-207; however, neither OGD nor EUK-207 produced significant changes in ERK1/2 and p38 phosphorylation. OGD-induced spectrin degradation was not modified by EUK-189 or EUK-207 in slices from p10 or 2-month-old rats, suggesting that their protective effects was not mediated through inhibition of calpain activation. Thus, both EUK-189 and EUK-207 provide neuroprotection in acute ischemic conditions, and this effect is related to elimination of free radical formation and partial reversal of ATP depletion, but not mediated by the activation or inhibition of the MEK/ERK or p38 pathways, or inhibition of calpain activation.     

Floral biology of the dioecious species Laurus nobilis L. (Lauraceae)

The present study examines the cytological, physiological, chemical and ecological characteristics of pollen and nectar offered by male and female flowers of the dioecious plant Laurus nobilis. The various phases of floral phenology and the insect pollinators were observed. We used cytological methods to determine anther, pollen and nectary structure. Nectar sugar composition was evaluated by HPLC. Pollen viability in time was compared with cytoplasmic and intine water content. Pollen presentation was found to be reversible by opening and closing of anther valves, determined by hydration of the mechanical layer of the anther. Pollen, covered by pollenkitt, was presented for dispersal for 3 consecutive days and during this time the intine and cytoplasm lost water and pollen viability diminished. At germination exine ruptured together with the outermost layer of the intine. Nectaries of male flowers were observed on the anther filament and on staminodes of female flowers. The nectar consisted almost entirely of sucrose and was more concentrated in male flowers. Secreted through stomata, nectar was presented in a thin layer. In the study area, the main pollinators (about half the total number of all visits) were hymenopterans. Pollen is of the recalcitrant type due to its high water content (>30%) but its viability is long-lasting because the intine is thick and stores water, keeping the cytoplasm of the vegetative cell hydrated and viable, and because anther valves may close under adverse conditions, protecting the pollen. Insects are attracted by male and female flowers similarly, males offer nectar and pollen, whilst females only nectar.     

Distinct roles of CysLT1 and CysLT2 receptors in oxygen glucose deprivation-induced PC12 cell death

Cysteinyl leukotrienes are involved in ischemic brain injury, and their receptors (CysLT(1) and CysLT(2)) have been cloned. To clarify which subtype mediates the ischemic neuronal injury, we performed permanent transfection to increase CysLT(1) and CysLT(2) receptor expressions in PC12 cells. Oxygen glucose deprivation (OGD)-induced cell death was detected by Hoechst 33258 and propidium iodide fluorescent staining as well as by flow cytometry. OGD induced late phase apoptosis mainly and necrosis minimally. Over-expression of CysLT(1) receptor decreased and over-expression of CysLT(2) receptor increased OGD-induced cell death. An agonist LTD(4) (10(-7)M) also induced apoptosis, especially in CysLT(2) receptor over-expressing cells. A selective CysLT(1) receptor antagonist montelukast did not affect OGD-induced apoptosis; while non-selective CysLT receptor antagonist Bay u9773 inhibited OGD-induced apoptosis, especially in CysLT(2) receptor over-expressing cells. Thus, CysLT(1) and CysLT(2) receptors play distinct roles in OGD-induced PC12 cell death; CysLT(1) attenuates while CysLT(2) facilitates the cell death.     

Xylem cavitation and hydraulic control of stomatal conductance in Laurel (Laurus nobilis L.)

The possible link between stomatal conductance (g_L), leaf water potential ( Ψ _L) and xylem cavitation was studied in leaves and shoots of detached branches as well as of whole plants of Laurus nobilis L. (Laurel). Shoot cavitation induced complete stomatal closure in air‐dehydrated detached branches in less than 10 min. By contrast, a fine regulation of g_L in whole plants was the consequence of Ψ _L reaching the cavitation threshold ( Ψ _CAV) for shoots. A pulse of xylem cavitation in the shoots was paralleled by a decrease in g_L of about 50%, while Ψ _L stabilized at values preventing further xylem cavitation. In these experiments, no root signals were likely to be sent to the leaves from the roots in response to soil dryness because branches were either detached or whole plants were growing in constantly wet soil. The stomatal response to increasing evaporative demand appeared therefore to be the result of hydraulic signals generated during shoot cavitation. A negative feedback link is proposed between g_L and Ψ _CAV rather than with Ψ _L itself.     

Notoginsenoside R1 protects oxygen and glucose deprivation-induced injury by upregulation of miR-21 in cardiomyocytes

Notoginsenoside R1 (NG-R1) is a major component of Panax notoginseng, which has been used clinically for the treatment of diabetic nephropathy for centuries in China. This study aimed to reveal the functional impacts and the underlying mechanisms of NG-R1 on oxygen-glucose deprivation (OGD)-injured cardiomyocytes. Rat cardiomyocyte line H9c2 and primary cardiomyocytes were subjected to OGD with or without NG-R1 treatment. The expression levels of miR-21 and phosphatase and tensin homolog (PTEN) in the cell were altered by microRNA, vector or short-hairpin RNA transfections. Thereafter, changes in cell viability, apoptosis, and PI3K/AKT signaling were monitored. NG-R1 with low concentrations had no impact on H9c2 cells viability, but 80 μM of NG-R1 significantly reduced cell viability. NG-R1 (20 μM) protected H9c2 cells and primary cardiomyocytes against OGD-induced cell damage, as cell viability was increased, apoptotic cell rate was reduced, and Bax, cleaved caspase-3 and -9 were downregulated by addition of NG-R1. MiR-21 was low expressed in response to OGD exposure, while was highly expressed by NG-R1 treatment. PTEN was a direct target of miR-21. More interestingly, OGD-induced cell damage could be recovered by miR-21 overexpression or PTEN silence. Furthermore, PTEN silence recovered OGD-blocked PI3K/AKT signaling pathway. To conclude, this study demonstrated that NG-R1 exerted remarkable benefits in reduction of OGD-induced cardiomyocyte loss. The cardioprotective actions of NG-R1 possibly via upregulation of miR-21, repressing the expression of miR-21's target PTEN and thereby preventing the blockage of PI3K/AKT signaling pathway.     

Inhibition of NGF deprivation-induced death by low oxygen involves suppression of BIMEL and activation of HIF-1

Changes in O(2) tension can significantly impact cell survival, yet the mechanisms underlying these effects are not well understood. Here, we report that maintaining sympathetic neurons under low O(2) inhibits apoptosis caused by NGF deprivation. Low O(2) exposure blocked cytochrome c release after NGF withdrawal, in part by suppressing the up-regulation of BIM(EL). Forced BIM(EL) expression removed the block to cytochrome c release but did not prevent protection by low O(2). Exposing neurons to low O(2) also activated hypoxia-inducible factor (HIF) and expression of a stabilized form of HIF-1alpha (HIF-1alpha(PP-->AG)) inhibited cell death in normoxic, NGF-deprived cells. Targeted deletion of HIF-1alpha partially suppressed the protective effect of low O(2), whereas deletion of HIF-1alpha combined with forced BIM(EL) expression completely reversed the ability of low O(2) to inhibit cell death. These data suggest a new model for how O(2) tension can influence apoptotic events that underlie trophic factor deprivation-induced cell death.     

Ultrasound-targeted microbubble destruction (UTMD)-mediated miR-150-5p attenuates oxygen and glucose deprivation-induced cardiomyocyte injury by inhibiting TTC5 expression

Molecular Biology Reports - Cardiomyocyte injury is a typical feature in cardiovascular diseases. Changes in cardiomyocytes strongly affect the progression of cardiovascular diseases. This work...     

Neuroprotective effects of hyperbaric oxygen (HBO) therapy on neuronal death induced by sciatic nerve transection in rat

Background

Recent studies shows that hyperbaric oxygen (HBO) therapy exerts some protective effects against neural injuries. The purpose of this study was to determine the neuroprotective effects of HBO following sciatic nerve transection (SNT).

Methods

Rats were randomly divided into five groups (n?=?14 per group): Sham-operated (SH) group, SH?+?HBO group, SNT group, and SNT?+?pre- and SNT?+?post-HBO groups (100% oxygen at 2.0 atm absolute, 60 min/day for five consecutive days beginning on 1 day before and immediately after nerve transaction, respectively). Spinal cord segments of the sciatic nerve and related dorsal root ganglions (DRGs) were removed 4 weeks after nerve transection for biochemical assessment of malodialdehyde (MDA) levels in spinal cord, biochemical assessment of superoxide dismutase (SOD) and catalse (CAT) activities in spinal cord, immunohistochemistry of caspase-3, cyclooxigenase-2 (COX-2), S100beta (S100ß), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in spinal cord and DRG.

Results

The results revealed that MDA levels were significantly decreased in the SNT?+?pre-HBO group, while SOD and CAT activities were significantly increased in SNT?+?pre- and SNT?+?post-HBO treated rats. Attenuated caspase-3 and COX-2 expression, and TUNEL reaction could be significantly detected in the HBO-treated rats after nerve transection. Also, HBO significantly increased S100ß expression.

Conclusions

Based on these results, we can conclude that pre- and post-HBO therapy had neuroprotective effects against sciatic nerve transection-induced degeneration.

     

Short- and long-term modulation of the lutein epoxide and violaxanthin cycles in two species of the Lauraceae: sweet bay laurel (Laurus nobilis L.) and avocado (Persea americana Mill.)

Short- and long-term responses of the violaxanthin (V) and lutein epoxide (Lx) cycles were studied in two species of Lauraceae: sweet bay laurel (Laurus nobilis L.) and avocado (Persea americana L.). The Lx content exceeded the V content in shade leaves of both species. Both Lx and V were de-epoxidised on illumination, but only V was fully restored by epoxidation in low light. Violaxanthin was preferentially de-epoxidised in low light in L. nobilis. This suggests that Lx accumulates with leaf ageing, partly because its conversion to lutein is limited in shade. After exposure to strong light, shade leaves of avocado readjusted the total pools of alpha- and beta-xanthophyll cycles by de novo synthesis of antheraxanthin, zeaxanthin and lutein. This occurred in parallel with a sustained depression of F(v)/F(m). In Persea indica, a closely related but low Lx species, F(v)/F(m) recovered faster after a similar light treatment, suggesting the involvement of the Lx cycle in sustained energy dissipation. Furthermore, the seasonal correlation between non-reversible Lx and V photoconversions and pre-dawn F(v)/F(m) in sun leaves of sweet bay supported the conclusion that the Lx cycle is involved in a slowly reversible downregulation of photosynthesis analogous to the V cycle.     

Antibacterial activity and mechanism of chloroform fraction from aqueous extract of mugwort leaves (Artemisia argyi L.) against Staphylococcus aureus

In this work, the antibacterial activity and mechanism of chloroform fraction obtained from aqueous extract of mugwort leaves against Staphylococcus aureus were investigated. The extract showed obvious antibacterial activity against S. aureus which the minimum inhibitory concentration and minimum bactericidal concentration were determined to be 3·0 and 6·0 mg ml⁻¹ respectively. The mechanism study suggested that the extract could destroy the integrity of the S. aureus cell walls and increase the permeability of cell membrane in a certain concentration, but it could not kill S. aureus in a short time. Instead, the extract could make bacteria in a state of apoptosis for a long time, interfere with the normal physiological metabolism of bacteria, and eventually make bacteria die, which was confirm by scanning electronic microscope.     

Cyclin-dependent kinase 5 (CDK5) and neuronal cell death

Many neurological disorders like Parkinson's and Alzheimer's disease, amyotrophic lateral sclerosis (ALS) or stroke have in common a definite loss of CNS neurons due to apoptotic or necrotic neuronal cell death. Previous studies suggested that proapoptotic stimuli may trigger an abortive and, therefore, eventually fatal cell cycle reentry in postmitotic neurons. Neuroprotective effects of small molecule inhibitors of cyclin-dependent kinases (CDKs), which are key regulators of cell cycle progression, support the cell cycle theory of neuronal apoptosis. However, growing evidence suggests that deregulated CDK5, which is not involved in cell cycle control, rather than cell cycle relevant members of the CDK family, promotes neuronal cell death. Here we summarize the current knowledge about the involvement of CDK5 in neuronal cell death and discuss possible up- or downstream partners of CDK5. Moreover, we discuss potential therapeutic options that might arise from the identification of CDK5 as an important upstream element of neuronal cell death cascades.     

Amelioration of neuronal cell death in a spontaneous obese rat model by dietary restriction through modulation of ubiquitin proteasome system

Dietary restriction (DR) has been shown to increase longevity, delay onset of aging, reduce DNA damage and oxidative stress and prevent age-related decline of neuronal activity. We previously reported the role of altered ubiquitin proteasome system (UPS) in the neuronal cell death in a spontaneous obese rat model (WNIN/Ob rat). In this study, we investigated the effect of DR on obesity-induced neuronal cell death in a rat model. Two groups of 40-day-old WNIN/Ob rats were either fed ad libitum (Ob) or pair-fed with lean. The lean phenotype of WNIN/Ob rats served as ad libitum control. These animals were maintained for 6.5 months on their respective diet regime. At the end of the study, cerebral cortex was collected and markers of UPS, endoplasmic reticulum (ER) stress and autophagy were analyzed by quantitative real-time polymerase chain reaction, immunoblotting and immunohistochemistry. Chymotrypsin-like activity of proteasome was assayed by the fluorimetric method. Apoptotic cells were analyzed by TUNEL assay. DR improved metabolic abnormalities in obese rats. Alterations in UPS (up-regulation of UCHL1, down-regulation of UCHL5, declined proteasomal activity), increased ER stress, declined autophagy and increased expression of α-synuclein, p53 and BAX were observed in obese rats and DR alleviated these changes in obese rats. Further, DR decreased TUNEL-positive cells. In conclusion, DR in obese rats could not only restore the metabolic abnormalities but also preserved neuronal health in the cerebral cortex by preventing alterations in the UPS.     

Chemical Constituents of the Fssential Oils from the Leaves of Laurus nobilis and Tendency in Changes of the Constituents Month by Month

The chemical constituents of the essential oils from the leaves of Laurus nobilis L., have been identified both by capillary GC-MS and fused silica capillary GC Kovats retention in,lex of components. From laurel leaf oil, 45 compounds have been identified, among which, 19 compounds such as 1,4-cineole, sabinene hydrate etc. have not been found in the leaf oil previously. This paper studies systematically the constituents of essential oil from dry, fresh leaves, and those of annual, perennial, different district, and reported the changes of the chemical constituents month by month. Results showed that the yield of essential oil and 1,8-cineole content is the highest in July.     

Induction of mitochondrial dysfunction by poly(ADP-ribose) polymer: Implication for neuronal cell death

Poly(ADP-ribose) polymerase-1 (PARP-1) mediates neuronal cell death in a variety of pathological conditions involving severe DNA damage. Poly(ADP-ribose) (PAR) polymer is a product synthesized by PARP-1. Previous studies suggest that PAR polymer heralds mitochondrial apoptosis-inducing factor (AIF) release and thereby, signals neuronal cell death. However, the details of the effects of PAR polymer on mitochondria remain to be elucidated. Here we report the effects of PAR polymer on mitochondria in cells in situ and isolated brain mitochondria in vitro. We found that PAR polymer causes depolarization of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore early after injury. Furthermore, PAR polymer specifically induces AIF release, but not cytochrome c from isolated brain mitochondria. These data suggest PAR polymer as an endogenous mitochondrial toxin and will further our understanding of the PARP-1-dependent neuronal cell death paradigm.