Hypoxic Preconditioning Group Research Articles

Hypoxia preconditioning increases Notch1 activity by regulating DNA methylation in vitro and in vivo.

Our previous research has demonstrated that hypoxic preconditioning (HPC) can improve spatial learning and memory abilities in adult mice. Adult hippocampal neurogenesis has been associated with learning and memory. The Neurogenic locus notch homolog protein (Notch) was involved in adult hippocampal neurogenesis, as well as in learning and memory. It is currently unclear whether the Notch pathway regulates hippocampal neuroregeneration by modifying the DNA methylation status of the Notch gene following HPC. The HPC animal model and cell model were established through repeated hypoxia exposure using mice and the mouse hippocampal neuronal cell line HT22. Step-down test was conducted on HPC mice. Real-time PCR and Western blot analysis were used to assess the mRNA and protein expression levels of Notch1 and hairy and enhancer of split1 (HES1). The presence of BrdU-positive cells and Notch1 expression in the hippocampal dental gyrus (DG) were examined with confocal microscopy. The methylation status of the Notch1 was analyzed using methylation-specific PCR (MS-PCR). HT22 cells were employed to elucidate the impact of HPC on Notch1 in vitro. HPC significantly improved the step-down test performance of mice with elevated levels of mRNA and protein expression of Notch1 and HES1 (P < 0.05). The intensities of the Notch1 signal in the control group, the H group and the HPC group were 2.62 ± 0.57 × 107, 2.87 ± 0.84 × 107, and 3.32 ± 0.14 × 107, respectively, and the number of BrdU (+) cells in the hippocampal DG were 1.83 ± 0.54, 3.71 ± 0.64, and 7.29 ± 0.68 respectively. Compared with that in C and H group, the intensity of the Notch1 signal and the number of BrdU (+) cells increased significantly in HPC group (P < 0.05). The methylation levels of the Notch1 promoter 0.82 ± 0.03, 0.65 ± 0.03, and 0.60 ± 0.02 in the C, H, and HPC groups, respectively. The methylation levels of Notch1 decreased significantly (P < 0.05). The effect of HPC on HT22 cells exhibited similarities to that observed in the hippocampus. HPC may confer neuroprotection by activating the Notch1 signaling pathway and regulating its methylation level, resulting in the regeneration of hippocampal neurons.

Metabolomic and Proteomic Identification of Serum Exosome for Hypoxic Preconditioning Participants.

In high-altitude areas, hypoxic stress can elicit a series of physiological responses in humans. Exosomes play important roles in both local and distal cellular communications. We used ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) studies to analyze the differentially expressed metabolomics and proteomics in serum exosome of hypoxic preconditioning participants and control subjects in the hypoxic conditions. Fifty-seven military personnel were divided into hypoxic preconditioning group (n = 27) and control group (n = 30). One hundred thirty-six differentially expressed serum exosomal metabolites were found between the hypoxic preconditioning and control groups in the hypoxic conditions, and these differentially expressed metabolites were enriched in pathways related to lysine degradation, butanoate metabolism, GABAergic synapse, histidine metabolism, and linoleic acid metabolism. In addition, hypoxic preconditioning participants showed 102 excellent differential expressions of proteomics compared to controls, which involved actin cytoskeleton organization, hemostasis, complement and coagulation cascades, vesicle-medicated transport, wound healing, etc. We revealed that the expression of exosomal metabolites and proteomics in hypoxic preconditioning participants was significantly different compared to controls in hypoxic conditions.

Hypoxic Preconditioning Ameliorates Amyloid-β Pathology and Longterm Cognitive Decline in AβPP/PS1 Transgenic Mice.

Hypoxic Preconditioning (HPC) has been well established to trigger endogenous mechanisms of neuroprotection basing on models of hypoxic and ischemic diseases in the Central Nervous System (CNS). However, its effects against Alzheimer's Disease (AD) still lack substantial evidence and in-depth exploration. The present study aimed to investigate the impacts of HPC on AD-related memory decline and amyloid-β (Aβ) pathology in AβPP/PS1 transgenic mice. Seven-week-old AβPP/PS1 transgenic mice were randomized into HPC and non-HPC groups. The HPC groups were treated with early and repetitive HPC for four weeks, while the non-HPC group was raised under normoxia condition. All the animals were then raised until the age of 28 weeks when Morris water maze tests were conducted to examine the animals' spatial memory. Indicators for Aβ pathology (soluble Aβ levels and numbers of Aβ plaques) and the expression of relevant proteins were measured to explore potential mechanisms. The results showed that HPC ameliorated memory decline and Aβ pathology in AβPP/PS1 mice. The protein levels of Amyloid-β Precursor Protein (AβPP) and β-site APP Cleaving Enzyme 1 (BACE1) were reduced while that of Hypoxic inducible factor 1α (HIF-1α) was elevated in HPC groups. HPC might be a promising strategy for AD intervention. Its potential protection might be realized via downregulating the expressions of AβPP and BACE1 and hence inhibiting Aβ pathology. Notably, HIF-1α might play a key role in mediating subsequent neuroadaptive changes following HPC.

UHPLC-QTOFMS-Based Metabolomic Analysis of the Hippocampus in Hypoxia Preconditioned Mouse

Background: Hypoxia appears in a number of extreme environments, including high altitudes, the deep sea, and during aviation, and occurs in cancer, cardiovascular disease, respiratory failures and neurological disorders. Though it is well recognized that hypoxic preconditioning (HPC) exerts endogenous neuroprotective effect against severe hypoxia, the mediators and underlying molecular mechanism for the protective effect are still not fully understood. This study established a hippocampus metabolomics approach to explore the alterations associated with HPC.Methods: In this study, an animal model of HPC was established by exposing the adult BALB/c mice to acute repetitive hypoxia four times. Ultra-high liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOFMS) in combination with univariate and multivariate statistical analyses was employed to deciphering metabolic changes associated with HPC in hippocampus tissue. MetaboAnalyst 3.0 was used to construct HPC related metabolic pathways.Results: The significant metabolic differences in hippocampus between the HPC groups and control were observed, indicating that HPC mouse model was successfully established and HPC could caused significant metabolic changes. Several key metabolic pathways were found to be acutely perturbed, including phenylalanine, tyrosine and tryptophan biosynthesis, taurine and hypotaurine metabolism, phenylalanine metabolism, glutathione metabolism, alanine, aspartate and glutamate metabolism, tyrosine metabolism, tryptophan metabolism, purine metabolism, citrate cycle, and glycerophospholipid metabolism.Conclusion: The results of the present study provided novel insights into the mechanisms involved in the acclimatization of organisms to hypoxia, and demonstrated the neuroprotective mechanism of HPC.

Paclitaxel enhances the protective effect of myocardial ischemia preconditioning on ischemia/reperfusion injury in aged rat

Objective: To investigate if paclitaxel can enhance the protective effect of myocardial ischemia preconditioning on ischemia/reperfusion injury in aged ratand explore related mechanism. Methods: Primary cardiomyocytes of Sprague-Dawley rats were isolated by trypsin and divided into 5 groups(n=6 each): control group, hypoxia injury group, hypoxia preconditioning group, paclitaxel group,and paclitaxel+hypoxia preconditioning group. The structure of microtubules and the expression of hypoxia-inducible factor-1α(HIF-1α) were analyzed by immunofluorescence staining. The Langendorff isolated heart perfusion model was applied in 4 groups: hypoxia reperfusion injury group, hypoxia preconditioning group, paclitaxel group, and paclitaxel+hypoxia preconditioning group. Each group was further divided into elderly subgroup and adult subgroup (n=6 each). Left ventricular developed pressure and maximum rate of rise in left ventricular pressure were analyzed. Results: (1) Primary cardiomyocyte experiments showed that the myocardial tubular microtubule structure in control group was intact and evenly stained; most of the microtubules in the hypoxia-injured group were absent and the tubular tissue was broken; the hypoxia-induced damage on microtubule structure was smaller in the hypoxic preconditioning group compared with the hypoxic injury group (microtubule staining was not uniform, and the lattice structure was broken, but not that obvious as in the hypoxia group); the tubular structure of the microtubules of the paclitaxel group was basically complete, and the staining was basically uniform.The integrity of tubular structure was maintained to some extent, similar to a normal microtubule structure in paclitaxel+hypoxia preconditioning group. The expression of HIF-1α in the cytoplasm and nucleus was very low in the control group, which was evidenced in both cytoplasm and nucleus in the hypoxic injury group.The expression was further increased in hypoxic preconditioning group, significant nuclear HIF-1 expression was found in the paclitaxel group, the expression was aggregated in the nucleus in the Paclitaxel+ hypoxia preconditioning group. (2)In Langendorff isolated heart perfusion model, left ventricular developed pressure was similar between the elderly subgroup and the adult subgroup at the end of the infusion,after precondition, 5 minutes of reperfusion, 30 minutes of reperfusion, and 60 minutes of reperfusion in the hypoxic injury group (all P> 0.05).In the hypoxic injury group, both the elderly subgroup and the adult subgroup had lower left ventricular developed pressure at 30 minutes of reperfusion when compared with the end of the infusion((15.63±4.88) mmHg (1 mmHg=0.133 kPa) vs. (95.63±22.14)mmHg and (17.31±2.75)mmHg vs. (91.00±9.58)mmHg, respectively,all P<0.05). In the hypoxic preconditioning group, the adult subgroup had higher left ventricular developed pressure at 5 and 30 minutes of reperfusion when compared with the elderly subgroup((7.13±1.02) mmHg vs. (3.75±1.06)mmHg and (43.94±3.21)mmHg vs.(16.31±1.54)mmHg, respectively,all P<0.01). In the paclitaxel group, the adult subgroup had higher left ventricular developed pressure at 30 and 60 minutes of reperfusion when compared with the elderly subgroup((44.31±7.59)mmHg vs. (5.44±1.21)mmHg, (51.56±6.03)mmHg vs. (22.19±5.14)mmHg, respectively, all P<0.01). In the paclitaxel+hypoxia preconditioning group, both the elderly subgroup and the adult subgroup had lower left ventricular developed pressure at 30 minutes of reperfusion when compared with the end of the infusion((18.63±4.30)mmHg vs. (99.94±8.23) mmHg, P<0.01; (49.69±5.34)mmHg vs. (95.31±5.26)mmHg, P<0.05). Meanwhile, the adult subgroup had higher left ventricular developed pressure at 30 minutes of reperfusion when compared with the elderly subgroup((49.69±5.34)mmHg vs. (18.63±4.33)mmHg, P<0.01).The adult subgroup had higher change rate of maximum rate of rise in left ventricular pressure at 60 minutes of reperfusion when compared with the elderly subgroup in hypoxia preconditioning group, paclitaxel group, and paclitaxel combined hypoxia preconditioning group((62.83±3.92)% vs. (33.33±3.20)%, (44.17±2.32)% vs. (36.67±2.88)%, (72.50±2.66)% vs. (53.17±2.56)%, respectively,all P<0.01). Conclusion: Paclitaxel can enhance the myocardial protective effect of myocardial ischemia preconditioning through stabilizing microtubules of cardiomyocytes and promoting HIF-1α localization in the nucleus.

Hypoxic preconditioning enhances cell hypoxia tolerance and correlated lncRNA and mRNA analysis

AimsHypoxia makes cells death but it is not always true. Short term anoxia can boost remaining cells activities and promote tissue repair. This study is to explore the effects of hypoxic preconditioning (HP) on osteoblasts and analyze the involved lncRNA and mRNA. Main methodsHuman osteoblast was used. Normoxic control group (N) were cultured in normoxic culture medium (L-DMEM, 10% FBS and 1% penicillin–streptomycin) 8 h. Long-term hypoxic group (H) were cultured in 4 h hypoxic culture medium (200 μmol/L CoCl2 and L-DMEM)/4 h normoxic culture medium. Hypoxic preconditioning group (HP) were received 3 times HP (cultured in 10 min hypoxic culture medium/10 min normoxic culture medium, which was HP 1times) and then cultured in 4 h hypoxic culture medium/4 h normoxic culture medium. Cell proliferation ability, osteonectin, transforming growth factor-β1, insulin like growth factors-1 and osteopontin were test. The lncRNA and mRNA was detected by high-throughput sequencing. p-Value < 0.05 was accepted as statistically significant. Key findingsCell proliferation activity: N > HP > P. Corrected osteonectin, TGF-β1, IGF-1 and osteopontin: HP > P > N. High-throughput sequencing of gene shown the expression of 4630 RNAs. Further, we identified that HP group made 239 and 160 target genes up-regulated and down-regulated respectively compared with H group (p < 0.05). GO, KEGG and PPI analysis indicated relevant biological processes. SignificanceHypoxic preconditioning alleviated cells anoxia tolerance and promoted cells to synthesis many cytokines. The protection mechanism involved many lncRNAs and mRNAs.

Hypoxic Preconditioning Protects SH-SY5Y Cell against Oxidative Stress through Activation of Autophagy

Oxidative stress plays a role in many neurological diseases. Hypoxic preconditioning (HPC) has been proposed as an intervention that protects neurons from damage by altering their response to oxidative stress. The aim of this study was to investigate the mechanisms by which HPC results in neuroprotection in cultured SH-SY5Y cells subjected to oxidative stress to provide a guide for future investigation and targeted interventions. SH-SY5Y cells were subjected to HPC protocols or control conditions. Oxidative stress was induced by H2O2. Cell viability was determined via adenosine triphosphate assay. Rapamycin and 3-methyxanthine (3-MA) were used to induce and inhibit autophagy, respectively. Monodansylcadaverine staining was used to observe the formation of autophagosomes. Levels of Microtubule-associated protein light chain 3 B (LC3B), Beclin 1, and p53 were measured by Western blot. Reactive oxygen species (ROS) were also determined. Cell viability in the HPC group following 24-h exposure to 600 μM H2O2 was 65.04 ± 12.91% versus 33.14 ± 5.55% in the control group. LC3B, Beclin 1, and autophagosomes were increased in the HPC group compared with controls. Rapamycin mimicked the protection and 3-MA decreased the protection. There was a moderate increase in ROS after HPC, but rapamycin can abolish the increase and 3-MA can enhance the increase. p53 accumulated in a manner consistent with cell death, and HPC-treated cells showed reduced accumulation of p53 as compared with controls. Treatment with rapamycin decreased p53 accumulation, and 3-MA inhibited the decrease in p53 induced by HPC. HPC protects against oxidative stress in SH-SY5Y cells. Mechanisms of protection may involve the activation of autophagy induced by ROS generated from HPC and the following decline in p53 level caused by activated autophagy in oxidative stress state. This is in line with recent findings in nonneuronal cell populations and may represent an important advance in understanding how HPC protects neurons from oxidative stress.

The roles of autophagy and PI3K-Akt-mTOR signaling transduction pathway in protection of high-glucose cultured cardiomyocytes against hypoxia/reoxygenation injury by hypoxia preconditioning

Objective To assess the roles of autophagy and phosphatidylinositol 3 hydroxy kinase(PI3K)-protein kinase B(Akt)-mammalian target of rapamycin(mTOR) signaling transduction pathway in protection of high-glucose cultured cardiomyocyte against anoxia/reoxygenation (AR) injury by hypoxia preconditioning (HPC). Methods Ccardiomyocytes cultured in high-glucose medium for 72 h were randomly divided into 5 groups: high glucose control group (S group), AR injury control group (AR group), HPC group, wortmannin+HPC group (Wo+HPC group), rapamycin+HPC group. Lactate dehydrogenase (LDH) leakage and cardiomyocyte apoptosis were analyzed. Microtubulesas sociated protein light(LC3)-Ⅱ, Beclin-1, PI3K, mTOR and phospho(p)-Akt/Akt ratio in cardiomyocytes were assessed by Western-blot assay. Results Compared with AR group, Wo+HPC group showed decrease in the leakage rate of LDH, the percentages of early and late apoptosis, and the levels of LC3-Ⅱ and Beclin-1 (P 0.05). Compared with the HPC group, Wo+HPC group displayed significant decreases in the leakage rate of LDH, the percentages of early and late apoptosis, and the levels of Beclin-1(P<0.05), but elevation of mTOR and PI3K, and p-Akt/Akt ratio(P<0.05). Conclusions Activation of PI3K-Akt-mTOR signaling transduction pathway and inhibition of autophagy can significantly improve the protection of high-glucose cultured cardiomyocytes against AR injury by HPC. Key words: Cardiomyocytes; High-glucose culture; Anoxia/reoxygenation injury; Hypoxia preconditioning; Autophagy; Phosphatidylinositol 3 hydroxy kinase-protein kinase B-mammalian target of rapamycin signaling transduction pathway

Hypoxia preconditioning protects Ca2+-ATPase activation of intestinal mucosal cells against R/I injury in a rat liver transplantation model.

AIMTo investigate the effect of ischaemia and reperfusion (I/R) injury on the Ca2+-ATPase activation in the intestinal tissue of a rat autologous orthotopic liver transplantation model and to determine if hypoxia preconditioning (HP) therapy induces HIF-1α to protect rat intestinal tissue against I/R injury.METHODSRats received non-lethal hypoxic preconditioning therapy to induce HIF-1α expression. We used an autologous orthotopic liver transplantation model to imitate the I/R injury in intestinal tissue. Then, we detected the microstructure changes in small intestinal tissues, Ca2+-ATPase activity, apoptosis, and inflammation within 48 h postoperatively.RESULTSHIF-1α expression was significantly increased in intestinal tissue at 12 h postoperatively in rats that were exposed to a hypoxic environment for 90 min compared with a non-HP group (HP vs AT, P = 0.0177). Pathological analysis was performed on the intestinal mucosa cells, and the cells in the HP group appeared healthier than the cells in the AT group. The Ca2+-ATPase activity in the small intestinal cells in the AT group was significantly lower after the operation, and the Ca2+-ATPase activity in the HP group recovered faster than that in the AT group at 6 h postoperatively (HP vs AT, P = 0.0106). BCL-2 expression in the HP group was significantly higher than that in the AT group at 12 h postoperatively (HP vs AT P = 0.0010). The expression of the inflammatory factors NO, SOD, IL-6, and TNF-α was significantly lower in the HP group than in the AT group.CONCLUSIONHypoxia-induced HIF-1α could protect intestinal mucosal cells against mitochondrial damage after I/R injury. HP could improve hypoxia tolerance in small intestinal mucosal cells and increase Ca2+-ATPase activity to reduce the apoptosis of and pathological damage to intestinal cells. HP could be a useful way to promote the earlier recovery of intestinal function after graft procedure.

Hypoxia preconditioning promotes cardiac stem cell survival and cardiogenic differentiation in vitro involving activation of the HIF-1\u03b1/apelin/APJ axis

BackgroundCardiac stem cells (CSCs) transplantation has been regarded as an optimal therapeutic approach for cardiovascular disease. However, inferior survival and low differentiation efficiency of these cells in the local infarct site reduce their therapeutic efficacy. In this study, we investigated the influence of hypoxia preconditioning (HP) on CSCs survival and cardiogenic differentiation in vitro and explored the relevant mechanism.MethodsCSCs were obtained from Sprague–Dawley rats and cells of the third passage were cultured in vitro and exposed to hypoxia (1% O2). Cells survival and apoptosis were evaluated by MTS assay and flow cytometry respectively. Cardiogenic differentiation was induced by using 5-azacytidine for another 24 h after the cells experienced HP. Normoxia (20% O2) was used as a negative control during the whole process. Cardiogenic differentiation was assessed 2 weeks after the induction. Relevant molecules were examined after HP and during the differentiation process. Anti-hypoxia-inducible factor-1α (HIF-1α) small interfering RNA (siRNA), anti-apelin siRNA, and anti-putative receptor protein related to the angiotensin receptor AT1 (APJ) siRNA were transfected in order to block their expression, and relevant downstream molecules were detected.ResultsCompared with the normoxia group, the hypoxia group presented more rapid growth at time points of 12 and 24 h (p < 0.01). Cells exhibited the highest proliferation rate at the time point of 24 h (p < 0.01). The cell apoptosis rate significantly declined after 24 h of hypoxia exposure (p < 0.01). Expression levels of HIF-1α, apelin, and APJ were all enhanced after HP. The percentage of apelin, α-SA, and cTnT positive cells was greatly increased in the HP group after 2 weeks of induction. The protein level of α-SA and cTnT was also significantly elevated at 7 and 14 days (p < 0.01). HIF-1α, apelin, and APJ were all increased at different time points during the cardiogenic differentiation process (p < 0.01). Knockdown of HIF-1α, apelin or APJ by siRNAs resulted in a significant reduction of α-SA and cTnT. HIF-1α blockage caused a remarkable decrease of apelin and APJ (p < 0.01). Expression levels of apelin and APJ were depressed after the inhibition of apelin (p < 0.01).ConclusionHP could effectively promote CSCs survival and cardiogenic differentiation in vitro, and this procedure involved activation of the HIF-1α/apelin/APJ axis. This study provided a new perspective for exploring novel strategies to enhance CSCs transplantation efficiency.

UPLC‑QTOFMS‑based metabolomic analysis of the serum of hypoxic preconditioning mice.

Hypoxic preconditioning (HPC) is well-known to exert a protective effect against hypoxic injury; however, the underlying molecular mechanism remains unclear. The present study utilized a serum metabolomics approach to detect the alterations associated with HPC. In the present study, an animal model of HPC was established by exposing adult BALB/c mice to acute repetitive hypoxia four times. The serum samples were collected by orbital blood sampling. Metabolite profiling was performed using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS), in conjunction with univariate and multivariate statistical analyses. The results of the present study confirmed that the HPC mouse model was established and refined, suggesting significant differences between the control and HPC groups at the molecular levels. HPC caused significant metabolic alterations, as represented by the significant upregulation of valine, methionine, tyrosine, isoleucine, phenylalanine, lysophosphatidylcholine (LysoPC; 16:1), LysoPC (22:6), linoelaidylcarnitine, palmitoylcarnitine, octadecenoylcarnitine, taurine, arachidonic acid, linoleic acid, oleic acid and palmitic acid, and the downregulation of acetylcarnitine, malate, citrate and succinate. Using MetaboAnalyst 3.0, a number of key metabolic pathways were observed to be acutely perturbed, including valine, leucine and isoleucine biosynthesis, in addition to taurine, hypotaurine, phenylalanine, linoleic acid and arachidonic acid metabolism. The results of the present study provided novel insights into the mechanisms involved in the acclimatization of organisms to hypoxia, and demonstrated the protective mechanism of HPC.

Hypoxic Preconditioning Augments the Therapeutic Efficacy of Bone Marrow Stromal Cells in a Rat Ischemic Stroke Model.

Transplantation of bone marrow stromal cells (BMSCs) is a promising therapy for ischemic stroke, but the poor oxygen environment in brain lesions limits the efficacy of cell-based therapies. Here, we tested whether hypoxic preconditioning (HP) could augment the efficacy of BMSC transplantation in a rat ischemic stroke model and investigated the underlying mechanism of the effect of HP. In vitro, BMSCs were divided into five passage (P0, P1, P2, P3, and P4) groups, and HP was applied to the groups by incubating the cells with 1% oxygen for 0, 4, 8, 12, and 24h, respectively. We demonstrated that the expression of hypoxia-inducible factor-1α (HIF-1α) was increased in the HP-treated BMSCs, while their viability was unchanged. We also found that HP decreased the apoptosis of BMSCs during subsequent simulated ischemia-reperfusion (I/R) injury, especially in the 8-h HP group. In vivo, a rat transient focal cerebral ischemia model was established. These rats were administered normal cultured BMSCs (N-BMSCs), HP-treated BMSCs (H-BMSCs), or DMEM cell culture medium (control) at 24h after the ischemic insult. Compared with the DMEM control group, the two BMSC-transplanted groups exhibited significantly improved functional recovery and reduced infarct volume, especially the H-BMSC group. Moreover, HP decreased neuronal apoptosis and enhanced the expression of BDNF and VEGF in the ischemic brain. Survival and differentiation of transplanted BMSCs were also increased by HP, and the quantity of engrafted BMSCs was significantly correlated with neurological function improvement. These results suggest that HP may enhance the therapeutic efficacy of BMSCs in an ischemic stroke model. The underlying mechanism likely involves the inhibition of caspase-3 activation and an increasing expression of HIF-1α, which promotes angiogenesis and neurogenesis and thereby reduces neuronal death and improves neurological function.

ABSTRACTS 7th World Congress of Mountain & Wilderness Medicine A combined meeting of the International Society for Mountain Medicine and the Wilderness Medical Society July 30-August 4, 2016 Telluride, Colorado.

ABSTRACTS 7th World Congress of Mountain & Wilderness Medicine A combined meeting of the International Society for Mountain Medicine and the Wilderness Medical Society July 30-August 4, 2016 Telluride, Colorado.

Protective effect of hypoxic preconditioning on SH-SY5Y cells injured by oxygen-glucose deprivation

To investigate the protection effects of hypoxic preconditioning(HPC) on the SH-SY5Y cell injured by oxygen-glu-cose deprivation(OGD),and to discuss the possible mechanism. SH-SY5Y cells were randomly divided into 4 groups. In normal group,the cells were cultured without OGD treatment. In HPC group,the cultured SH-SY5Y cells were treated for 5 days by intermittently ex-posing to hypoxic gas mixture (2% O2,5% CO2) for 30 min in every day. In OGD group,the culture medium was replaced by glucose-free medium and the cells were transferred to a humidified incubation chamber flushed by a gas mixture of 1% O2 and 5% CO2 for 10 h. After that, the cells were fed with glucose-supplemented medium and cultured under normoxic condition for 24 h. In HPC+OGD group,the cultured SH-SY5Y cells were treated for 5 days by intermittently exposing to hypoxic gas mixture for 30 min in each day, then the cells were given the same treatments as those in OGD group. The cell viability was assessed by MTT assay. The degree of the cell damage was evaluated by deter-mining lactate dehydrogenase (LDH) leakage. TUNEL staining were used to detect the variation of cell apoptosis. The expression of Caspase 3 and hypoxia inducible factor-1α(HIF-1α) at protein levels was examined by Western blot. Hypoxic preconditioning relieved the cells apoptosis,decreased the amount of LDH leakage and improved the viability of SH-SY5Y cells injured by OGD (P<0.05). Western blot showed that the expression of Caspase 3 protein in HPC+OGD group was significantly lower than that in OGD group (P<0.05); HIF-1α protein expression was significantly higher than that of OGD group (P<0.05). Hypoxic preconditioning has protective effect on in vitro cultured SH-SY5Y cells injured by OGD. The mechanism may be related to the increase of HIF-1a protein.

Effects of hypoxic preconditioning on learning and memory in mice with cerebral ischemia-reperfusion injury and underlying mechanisms

Objective To investigate the effects of hypoxic preconditioning on learning and memory and the possible protective mechanism in mice with cerebral ischemia-reperfusion injury. Methods Healthy adult male Kunming mice were randomly divided into five groups by Random number table: normal group(N group), hypoxic preconditioning group (HPC group), sham operation group (C group), ischemia-reperfusion group(O group), hypoxic preconditioning and ischemia-reperfusion group(HPC+ O group). HPC+ O group were given hypoxic preconditioning before 24h of ischemia- reperfusion. The escape latency was detected by Morris water maze and the neuron apoptosis of CA1 area of hippocampal was determined by immunofluorescence technique. Results The escape latency in HPC+ O group on the second, third and fourth day of MWM was (39.92±4.52)s, (30.98±2.44)s, (19.69±4.27)s, and significantly lower than that in O group((54.35±3.66)s, (46.31±4.81)s, (36.81±3.86)s). Mice in HPC+ O spent longer time in the target quadrant than that in O group((36.44±5.33)% and(24.5±2.59)%, respectively, P<0.05). Immunofluorescence showed that the apoptotic ration of nerve cells in hippocampal CA1 was significantly lower than that in O group(1.17±0.14 and 1.35±0.14, P<0.05). Conclusion Hypoxic preconditioning can increase hippocampal CA1 neurons hypoxia tolerance of ischemia reperfusion injury in mice, and reduce the incidence of neural cell apoptosis. Key words: Hypoxic preconditioning; Ischemia-reperfusion; Learning and memory; Apoptosis

The Effect of Hypoxic Preconditioning on Induced Schwann Cells under Hypoxic Conditions.

ObjectOur objective was to explore the protective effects of hypoxic preconditioning on induced Schwann cells exposed to an environment with low concentrations of oxygen. It has been observed that hypoxic preconditioning of induced Schwann cells can promote axonal regeneration under low oxygen conditions.MethodRat bone marrow mesenchymal stem cells (MSCs) were differentiated into Schwann cells and divided into a normal oxygen control group, a hypoxia-preconditioning group and a hypoxia group. The ultrastructure of each of these groups of cells was observed by electron microscopy. In addition, flow cytometry was used to measure changes in mitochondrial membrane potential. Annexin V-FITC/PI staining was used to detect apoptosis, and Western blots were used to detect the expression of Bcl-2/Bax. Fluorescence microscopic observations of axonal growth in NG-108 cells under hypoxic conditions were also performed.ResultsThe hypoxia-preconditioning group maintained mitochondrial cell membrane and crista integrity, and these cells exhibited less edema than the hypoxia group. In addition, the cells in the hypoxia-preconditioning group were found to be in early stages of apoptosis, whereas cells from the hypoxia group were in the later stages of apoptosis. The hypoxia-preconditioning group also had higher levels of Bcl-2/Bax expression and longer NG-108 cell axons than were observed in the hypoxia group.ConclusionHypoxic preconditioning can improve the physiological state of Schwann cells in a severe hypoxia environment and improve the ability to promote neurite outgrowth.

UPLC-HRMS based metabolomics reveals the sphingolipids with long fatty chains and olefinic bonds up-regulated in metabolic pathway for hypoxia preconditioning

Hypoxia preconditioning (HPC) could protect cells, tissues, organs and systems from hypoxia injury, but the molecular mechanism still remained unclear. The ultra-high performance liquid chromatography coupled high resolution mass spectrometry (UPLC-HRMS) based metabolomics method was utilized to explore the key endogenous metabolites and metabolic pathways related to HPC. Our results clearly showed that the HPC mice model was established and refined, suggesting that there were significant differences between the control group and 6 × HPC group at the molecular levels. A serious of statistical analyses, including univariate analysis and multivariate analysis, were performed by the Progenesis QI software package and MetaboAnalyst web-server. The sphingolipid metabolic pathways were noticed due to the low p-value and high pathway impact calculated by the MetaboAnalyst and the pathways were altered under HPC condition. Especially, the sphingolipid compound sphingomyelin, ceramide, glucosylceramide, galactosylceramide and lactosylceramide were mapping in this metabolic pathway. Interestingly, these sphingolipid metabolites with olefinic bond in the long fatty chain were up-regulated, while those sphingolipids without olefinic bond were down-regulated. The results indicated that C24:1-Cers played a critical role in HPC and had potential in endogenous protective mechanism. Our data provided an insight to further reveal the protection mechanism of HPC.

The cardioprotective effect of hypoxic and ischemic preconditioning in dogs with myocardial ischemia-reperfusion injury using a double-bypass model.

The effects of preconditioning on cardioprotection have mainly been studied in vitro. No sufficient in vivo experiments have been performed to optimize ischemic preconditioning (IPC) or hypoxic preconditioning (HPC) for clinical applications. The purpose of this study was to establish a canine double-bypass model to examine the effect of IPC and HPC on cardiomyocytes and heart function. A double-bypass procedure to enable independent control of systemic and coronary circulation was established in dogs. The animals were divided into control, HPC, and IPC groups (n=6 each). Indicators of cardiac function, including cardiodynamics, hemodynamics, ATP, and cardiac troponin I (cTnI) levels; myocardium morphology; and myocardiocyte apoptosis were determined. Both IPC and HPC attenuated the reperfusion-induced decrease in left ventricular end systolic pressure seen in the control group. Both the HPC and IPC groups had lower serum cTnI levels, better myocardiocyte histology, and lower rates of apoptosis compared to the control group without preconditioning. HPC reduced the abnormal cardiomyocyte histology and apoptosis to a greater extent than IPC, and only HPC significantly restored the depletion of ATP. This study demonstrates the effectiveness of the double-bypass model for the optimized study of both HPC and IPC. The results suggest that HPC may provide better cardioprotection than IPC.

Proteomic analysis of the mouse brain after repetitive exposure to hypoxia

Hypoxic preconditioning (HPC) is known to have a protective effect against hypoxic damage; however, the precise mechanisms involved remain unknown. In this study, an acute and repetitive hypoxia mouse model, two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/TOF-MS), and Western blot experiments were used to identify the differential expression of key proteins in the mouse brain during HPC. Approximately 2100 2D-DIGE spots were observed following gel imaging and spot detection. Significant differences (p<0.05) in the expression of 66 proteins were observed between the 3× HPC treatment group and the control group, 45 proteins were observed between the 6× HPC treatment group and the control group, and 70 proteins were observed between the 3× HPC treatment group and the 6× HPC group. Consistent results among Western blot, 2D-DIGE and MS methods were observed for the proteins, ATP synthase subunit alpha, malate dehydrogenase, guanine nucleotide-binding protein subunit beta-1 and proteasome subunit alpha type-2. The proteins associated with ATP synthesis and the citric acid cycle were down-regulated, while those linked to glycolysis and oxygen-binding were up-regulated. This proteomic analysis of the mouse brain after HPC furthers understanding of the molecular pathways involved in the protective effect of HPC and these findings provide new insight into the mechanisms of hypoxia and HPC.

The effects of age and resveratrol on the hypoxic preconditioning protection against hypoxia-reperfusion injury: studies in rat hearts and human cardiomyocytes.

The loss of effectiveness of ischaemic preconditioning in protecting old hearts from ischaemia/reperfusion damage is thought to be due to low sirtuin 1 levels in old hearts. We sought to determine whether resveratrol (RES), an activator of sirtuin 1, would restore this protection to that seen with ischaemic preconditioning in young hearts. A Langendorff heart perfusion model was established in 80 old and 80 adult rats to test the effects of hypoxic preconditioning (HPC) and/or RES on preventing hypoxia-reperfusion (H/R) injury. The effects were further tested by comparing the effects of HPC and RES on cell survival rate and lactate dehydrogenase (LDH) in cardiomyocytes from 15 old and 15 young humans. The HPC + RES group performed better in both adult and old groups than the corresponding H/R, HPC and RES groups, causing ∼50% in the adult and 40% in the old group restoration of left ventricular developed pressure and ∼90% in the adult and 80% in the old group restoration of dp/dtmax. HPC and RES each reduced apoptosis in both groups. The HPC + RES treatment showed an additive benefit in reducing apoptosis in the adult group but not in the old group. In H/R-treated young and old human cardiomyocytes, cell survival and LDH level were significantly improved in the RES + HPC group compared with the HPC group. This study showed that RES lessened the ageing effect and enhanced the cardioprotective effect of HPC in older individuals.