Perspectives on Neonatal Hypoxia/Ischemia-Induced Edema Formation

Neonatal hypoxia/ischemia (HI) is the most common cause of developmental neurological, cognitive and behavioral deficits in children, with hyperoxia (HHI) treatment being a clinical therapy for newborn resuscitation. Although cerebral edema is a common outcome after HI, the mechanisms leading to excessive fluid accumulation in the brain are poorly understood. Given the rigid nature of the bone-encased brain matter, knowledge of edema formation in the brain as a consequence of any injury, as well as the importance of water clearance mechanisms and water and ion homeostasis is important to our understanding of its detrimental effects. Knowledge of the pathological process underlying the appearance of dysfunctional outcomes after development of cerebral edema after neonatal HI in the developing brain and the molecular events triggered will allow a rational assessment of HHI therapy for neonatal HI and determine whether this treatment is beneficial or harmful to the developing infant.     

Hypoxia Ischemia-Mediated Cell Death in Neonatal Rat Brain

The examination of Bcl-2-associated X protein (Bax) protein’s role in the activation of cognate nuclear, mitochondrial and ER cell death signaling cascades and the resulting effects on cell death phenotype in the brain after neonatal hypoxia-ischemia (HI) requires an understanding of neonatal HI insult and progression, as well as, its dysfunctional outcomes. In addition, knowledge of key concepts of oxidative stress, a major injurious component of HI, and the different cell death phenotypes (i.e. apoptosis and necrosis) will aid the design of appropriate useful experimental paradigms. Here we discuss organelle cell death signaling cascades in the context of the different cell death phenotypes associated with animal models of neonatal hypoxia ischemia and tissue culture models used in the study of hypoxia ischemia, focusing on the intracellular shifts of the Bcl-2 associated X protein (Bax) in the hypoxic brain. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Hypoxia Induces Synthesis of a Novel 22-kDa Protein in Neonatal Rat Oligodendrocytes

Neonatal (3-day-old) rat oligodendrocytes grown in monolayer culture and exposed to increasingly hypoxic culture conditions showed a dramatic reduction in myelin basic protein synthesis but no significant inhibition of Tran35S-label incorporation into oligodendrocyte proteins in general or into structural proteins such as actin. However, there was a dramatic increase in synthesis of a novel 22-kDa protein. Reoxygenation of cultures reversed the synthesis of the 22-kDa protein, and thiol and calpain protease inhibitors (EP-459 and leupeptin) did not prevent synthesis of the protein, suggesting that it did not result from proteolysis. The 22-kDa protein (which we have called hypoxin) was coimmunoprecipitated by a polyclonal antibody to actin but did not react with the anti-actin antibody on western blots. The synthesis of hypoxin accounted for up to 50% of the Tran35S-label incorporated into immunoprecipitated protein, suggesting that it plays a major role in the cell's response to hypoxia. Subcellular fractionation revealed that the 22-kDa protein was largely associated with the cytosolic/cytoskeletal compartment. However, it is unlikely to be one of the cytoskeleton-associated Rho or Rac low-molecular-mass (20-24 kDa) GTP-binding proteins because it did not bind [alpha-32P]GTP on western blots. Oligodendrocytes did not synthesize a 22-kDa protein in response to heat shock but did synthesize the typical 70- and 90-kDa heat-shock proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

不同缺氧时间制作新生大鼠脑室周围白质软化动物模型的比较

目的通过比较不同的缺氧时间,创建与人类早产儿脑室周围白质软化（periventricular leukomMacia,PVL）病理相似的可靠PVL动物模型。方法对2Et龄新生大鼠进行双侧颈总动脉结扎,分为缺氧2h组,缺氧1h组,缺氧0.5h组和缺氧0h组,另设Sham组。分别于术后2d比较各组动物死亡率,并进行大体和光镜下脑病理评估。结果缺氧大于0.5h有更高的动物死亡率（X^2=58.464,P〈0.01）,缺氧0.5h组和缺氧0h组在死亡率之间的差异无统计学意义（X^2=0.18,P=0.672）。脑大体病理显示,缺氧大于0.5h以液化坏死为主,缺氧0.5h和0h则以脑水肿或萎缩为主。光镜下脑病理显示,缺氧大于0.5h,其病变涉及灰质和白质。缺氧0.5h和0h,则主要造成白质损伤,皮质神经元损伤轻微。Sham组则无脑病理改变。结论对2日龄新生大鼠进行双侧颈总动脉结扎伴缺氧0.5h,创建PVL新生动物模型的效果最为理想。     

Transient Increase in Rab 3A and Synaptobrevin Immunoreactivity After Mild Hypoxia in Neonatal Rats

1. In the present work we describe the short term effects of mild neonatal hypoxia on the synapse as assessed by the immunoreactivity (IR) of twosynaptic proteins: rab 3A and synaptobrevin (VAMP).2. Using the sensitive methodology of immunoblotting, we measured rab 3A andVAMP-IR in homogenates from the cerebral cortex, hippocampus, and corpus striatum of control (breathing room air) and hypoxiated (breathing 95.5% N₂–6.5% O₂ for 70 min) 4-day-old rats at 1, 2, and 6 h after the end of the hypoxia. Immunostaining with examination by light microscopy was performed using the synaptic protein-specific antibodies on fixed brain sections from animals belonging to the same litter and submitted to hypoxia.3. A transient increase of VAMP-IR was observed in the hippocampus and corpus striatum, and for rab 3A in the striatum, 1 h after initiating reoxygenation.At the following time points the values returned to control levels. This effectwas less clearly observed in the immunostained sections.4. Mild hypoxia has an effect on sensitive brain regions, eliciting an increase in the IR of at least two proteins involved in the synaptic vesicle cycle. The transient nature of this effect possibly indicates the activation of endogenous neuroprotective mechanisms.     

Lycopene Protects against Hypoxia/Reoxygenation-Induced Apoptosis by Preventing Mitochondrial Dysfunction in Primary Neonatal Mouse Cardiomyocytes

Background

Hypoxia/reoxygenation(H/R)-induced apoptosis of cardiomyocytes plays an important role in myocardial injury. Lycopene is a potent antioxidant carotenoid that has been shown to have protective properties on cardiovascular system. The aim of the present study is to investigate the potential for lycopene to protect the cardiomyocytes exposed to H/R. Moreover, the effect on mitochondrial function upon lycopene exposure was assessed.

Methods and Findings

Primary cardiomyocytes were isolated from neonatal mouse and established an in vitro model of H/R which resembles ischemia/reperfusion in vivo. The pretreatment of cardiomyocytes with 5 µM lycopene significantly reduced the extent of apoptosis detected by TUNEL assays. To further study the mechanism underlying the benefits of lycopene, interactions between lycopene and the process of mitochondria-mediated apoptosis were examined. Lycopene pretreatment of cardiomyocytes suppressed the activation of the mitochondrial permeability transition pore (mPTP) by reducing the intracellular reactive oxygen species (ROS) levels and inhibiting the increase of malondialdehyde (MDA) levels caused by H/R. Moreover, the loss of mitochondrial membrane potential, a decline in cellular ATP levels, a reduction in the amount of cytochrome c translocated to the cytoplasm and caspase-3 activation were observed in lycopene-treated cultures.

Conclusion

The present results suggested that lycopene possesses great pharmacological potential in protecting against H/R-induced apoptosis. Importantly, the protective effects of lycopene may be attributed to its roles in improving mitochondrial function in H/R-treated cardiomyocytes.     

Changes in Levels of Purine and Pyrimidine Nucleotides During Acute Hypoxia and Recovery in Neonatal Rat Brain

Neonatal rat brains were examined for changes in levels of ATP, ADP, AMP, cyclic AMP, GTP, GDP, UTP, UDP, UMP, and CTP during exposure to 100% nitrogen for 20 min and subsequent recovery in air. During hypoxia, ATP, GTP, UTP, and CTP levels and the GTP/GDP ratio decreased to 38, 50, 26, 21, and 21%, respectively, of control levels. No significant change in cyclic AMP level was observed. The decrease in the total uridine nucleotide pool during hypoxia was markedly greater (to 53% of control levels) than that in the total adenine nucleotide pool (to 92% of control levels). During recovery, ATP and GTP levels were rapidly and almost completely restored. On the other hand, CTP levels returned slowly to control values after a 2-h recovery period. Restoration of the UTP level was slow and incomplete (87% of the control value even after a 3-h recovery period). The GTP/GDP ratio also did not return to normal. These data suggest that hypoxic insult to the neonate may have an effect on the synthesis of nucleotidyl sugars, phospholipids, and proteins in the brain, resulting in significant problems with developmental processes of the brain. The present study also showed that the delayed restorations of the UTP level and the GTP/GDP ratio were not seen in the brains of adult rats subjected to acute severe hypoxic insult.     

斑马缺硒病的血液细胞和血液生化指标测定

以某动物园中经临床观察、病理学检查、血液细胞和血液生化分析方面诊断为缺硒的4只斑马为研究对象,3只健康斑马作为对照,分析缺硒斑马血液细胞和血液生化检测值的变化,探讨斑马缺硒病的诊断方法。结果表明:缺硒病斑马的红细胞数量和血红蛋白量下降,中性粒细胞百分比上升而淋巴细胞百分比下降,血液生化指标分析显示乳酸脱氢酶、丙氨酸氨基转移酶、天门冬氨酸氨基转移酶、碱性磷酸酶和肌酸激酶活性升高,尿酸含量和谷胱甘肽过氧化物酶活力显著下降。这一结果在斑马缺硒病的诊断中具有重要意义。     

Lycopene Protects against Hypoxia/Reoxygenation Injury by Alleviating ER Stress Induced Apoptosis in Neonatal Mouse Cardiomyocytes

Endoplasmic reticulum (ER) stress induced apoptosis plays a pivotal role in myocardial ischemia/reperfusion (I/R)-injury. Inhibiting ER stress is a major therapeutic target/strategy in treating cardiovascular diseases. Our previous studies revealed that lycopene exhibits great pharmacological potential in protecting against the I/R-injury in vitro and vivo, but whether attenuation of ER stress (and) or ER stress-induced apoptosis contributes to the effects remains unclear. In the present study, using neonatal mouse cardiomyocytes to establish an in vitro model of hypoxia/reoxygenation (H/R) to mimic myocardium I/R in vivo, we aimed to explore the hypothesis that lycopene could alleviate the ER stress and ER stress-induced apoptosis in H/R-injury. We observed that lycopene alleviated the H/R injury as revealed by improving cell viability and reducing apoptosis, suppressed reactive oxygen species (ROS) generation and improved the phosphorylated AMPK expression, attenuated ER stress as evidenced by decreasing the expression of GRP78, ATF6 mRNA, sXbp-1 mRNA, eIF2α mRNA and eIF2α phosphorylation, alleviated ER stress-induced apoptosis as manifested by reducing CHOP/GADD153 expression, the ratio of Bax/Bcl-2, caspase-12 and caspase-3 activity in H/R-treated cardiomyocytes. Thapsigargin (TG) is a potent ER stress inducer and used to elicit ER stress of cardiomyocytes. Our results showed that lycopene was able to prevent TG-induced ER stress as reflected by attenuating the protein expression of GRP78 and CHOP/GADD153 compared to TG group, significantly improve TG-caused a loss of cell viability and decrease apoptosis in TG-treated cardiomyocytes. These results suggest that the protective effects of lycopene on H/R-injury are, at least in part, through alleviating ER stress and ER stress-induced apoptosis in neonatal mouse cardiomyocytes.     

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo “pre-conditioning” stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.     

引入西藏高原家兔的血清生化指标测定

目的了解和掌握引入西藏高原家兔的生理生化指标,以便为实践教学、临床诊疗和科学研究提供参数。方法采用朗道全自动生化分析仪对引入西藏高原20年的加利福尼亚兔(Californian rabbit)和中国白兔(Chinese white rabbit)的12项血清生化指标进行了测定。结果加利福尼亚兔在品种内比较发现TP、ALB、A/G、CRE、CHOL、LDH指标表现极强的雄兔特征(P0.01),而ALT、GLU测定值雌兔明显高于雄兔(P0.05);中国白兔在品种内比较发现CRE、LDH指标显示差异有显著性(P0.01),CRE指标是雄兔高于雌兔,LDH参数是雌兔高于雄兔。加利福尼亚雄兔与中国白雄兔的参数比较中发现AST、TP、ALB、GLO、A/G、GLU、CRE、LDH指标显示差异有显著性(P0.01),而TG、BUN指标显示差异有显著性(P0.05);加尼福利亚母兔与中国白母兔的参数比较中发现ALT、GLO、CRE、TG指标显示差异有显著性(P0.05),AST、TP、BUN、LDH指标显示差异有显著性(P0.01)。结论实验结果将为西藏高原的教学、临床诊疗和科学研究提供生理数据参考。     

A Lab Assembled Microcontroller-Based Sensor Module for Continuous Oxygen Measurement in Portable Hypoxia Chambers

Background

Hypoxia-based cell culture experiments are routine and essential components of in vitro cancer research. Most laboratories use low-cost portable modular chambers to achieve hypoxic conditions for cell cultures, where the sealed chambers are purged with a gas mixture of preset O₂ concentration. Studies are conducted under the assumption that hypoxia remains unaltered throughout the 48 to 72 hour duration of such experiments. Since these chambers lack any sensor or detection system to monitor gas-phase O₂, the cell-based data tend to be non-uniform due to the ad hoc nature of the experimental setup.

Methodology

With the availability of low-cost open-source microcontroller-based electronic project kits, it is now possible for researchers to program these with easy-to-use software, link them to sensors, and place them in basic scientific apparatus to monitor and record experimental parameters. We report here the design and construction of a small-footprint kit for continuous measurement and recording of O₂ concentration in modular hypoxia chambers. The low-cost assembly (US$135) consists of an Arduino-based microcontroller, data-logging freeware, and a factory pre-calibrated miniature O₂ sensor. A small, intuitive software program was written by the authors to control the data input and output. The basic nature of the kit will enable any student in biology with minimal experience in hobby-electronics to assemble the system and edit the program parameters to suit individual experimental conditions.

Results/Conclusions

We show the kit’s utility and stability of data output via a series of hypoxia experiments. The studies also demonstrated the critical need to monitor and adjust gas-phase O₂ concentration during hypoxia-based experiments to prevent experimental errors or failure due to partial loss of hypoxia. Thus, incorporating the sensor-microcontroller module to a portable hypoxia chamber provides a researcher a capability that was previously available only to labs with access to sophisticated (and expensive) cell culture incubators.     

3-Hydroxybutyrate Aids the Recovery of the Energy State from Aglycaemic Hypoxia of Adult but Not Neonatal Rat Brain Slices

Abstract: The level of phosphocreatine (PCr) and the intracellular pH (pH_i) of superfused cortical brain slices from adult or 10-day-old rats were monitored using ³¹P NMR. When the glucose in the superfusing medium was replaced by 3-hydroxybutyrate (3HB), there was a significant reduction in PCr of the adult but not the neonatal slices. The level of PCr of the adult slices was reduced by a greater amount by aglycaemic hypoxia compared with the neonatal brain slices and pH_i was decreased by the same amount. After aglycaemic hypoxia, the levels of PCr of the neonatal slices recovered to the same extent when perfused with glucose or 3HB alone or a mixture of glucose and 3HB. The recovery of the PCr was significantly more in the neonatal than the adult brain slices with glucose alone after aglycaemic hypoxia, whereas pH_i returned to control levels in both tissue types and with all substrates. The relative recovery of the PCr of the adult slices after aglycaemic hypoxia was the same with either 3HB or glucose. However, if glucose and 3HB were applied together, recovery of PCr was significantly improved compared with glucose alone.     

Hypoxia. Hypoxia in the pathogenesis of systemic sclerosis

Autoimmunity, microangiopathy and tissue fibrosis are hallmarks of systemic sclerosis (SSc). Vascular alterations and reduced capillary density decrease blood flow and impair tissue oxygenation in SSc. Oxygen supply is further reduced by accumulation of extracellular matrix (ECM), which increases diffusion distances from blood vessels to cells. Therefore, severe hypoxia is a characteristic feature of SSc and might contribute directly to the progression of the disease. Hypoxia stimulates the production of ECM proteins by SSc fibroblasts in a transforming growth factor-β-dependent manner. The induction of ECM proteins by hypoxia is mediated via hypoxia-inducible factor-1α-dependent and -independent pathways. Hypoxia may also aggravate vascular disease in SSc by perturbing vascular endothelial growth factor (VEGF) receptor signalling. Hypoxia is a potent inducer of VEGF and may cause chronic VEGF over-expression in SSc. Uncontrolled over-expression of VEGF has been shown to have deleterious effects on angiogenesis because it leads to the formation of chaotic vessels with decreased blood flow. Altogether, hypoxia might play a central role in pathogenesis of SSc by augmenting vascular disease and tissue fibrosis.     

当归对宫内缺氧新生大鼠大脑皮质神经元与VEGF mRNA表达的影响

目的 探讨宫内缺氧对新生大鼠大脑皮质神经元与VEGF mRNA表达的影响以及当归的调控作用.方法 孕14 d健康SD雌性大鼠15只,随机分为对照组、缺氧组和当归组各5只,于孕14 d开始将当归组与缺氧组孕鼠置于低张氧浓度三气培养箱中,制作胎鼠宫内缺氧模型,此前一小时按8 mL/kg分别给予当归和生理盐水尾静脉注射,对照组不缺氧,余同缺氧组.三组孕鼠分娩当日每窝随机选取新生鼠4只,取脑组织多聚甲醛固定,石蜡包埋切片、NSE mRNA、VEGF mRNA原位杂交,400倍拍照、IPP6.0软件图像分析.结果 缺氧组新生大鼠大脑皮质NSE mRNA阳性细胞数较对照组减少,积分光密度值(IOD)降低(P＜0.05),VEGF mRNA阳性细胞IOD值升高(P＜0.05);当归组新生大鼠大脑皮质NSE mRNA阳性细胞数较缺氧组增多、IOD值增高(P＜0.05),VEGF mRNA阳性细胞IOD值增高(P＜0.05).结论 宫内缺氧可致新生大鼠大脑皮质神经元受损,当归注射液对此损伤有一定保护作用,其机制可能是通过上调大脑皮质VEGF mRNA的表达而使缺氧环境改善.