TRPM2 channel: A novel target for alleviating ischaemia‐reperfusion,chronic cerebral hypo‐perfusion and neonatal hypoxic‐ischaemic brain damage

The transient receptor potential melastatin‐related 2 (TRPM2) channel, a reactive oxygen species (ROS)‐sensitive cation channel, has been well recognized for being an important and common mechanism that confers the susceptibility to ROS‐induced cell death. An elevated level of ROS is a salient feature of ischaemia‐reperfusion, chronic cerebral hypo‐perfusion and neonatal hypoxia‐ischaemia. The TRPM2 channel is expressed in hippocampus, cortex and striatum, the brain regions that are critical for cognitive functions. In this review, we examine the recent studies that combine pharmacological and/or genetic interventions with using in vitro and in vivo models to demonstrate a crucial role of the TRPM2 channel in brain damage by ischaemia‐reperfusion, chronic cerebral hypo‐perfusion and neonatal hypoxic‐ischaemia. We also discuss the current understanding of the underlying TRPM2‐dependent cellular and molecular mechanisms. These new findings lead to the hypothesis of targeting the TRPM2 channel as a potential novel therapeutic strategy to alleviate brain damage and cognitive dysfunction caused by these conditions.     

Irradiation to the immature brain attenuates neurogenesis and exacerbates subsequent hypoxic‐ischemic brain injury in the adult

Cranial radiotherapy is common in pediatric oncology. Our purpose was to investigate if irradiation (IR) to the immature brain would increase the susceptibility to hypoxic‐ischemic injury in adulthood. The left hemisphere of postnatal day 10 (P10) mice was irradiated with 8 Gy and subjected to hypoxia‐ischemia (HI) on P60. Brain injury, neurogenesis and inflammation were evaluated 30 days after HI. IR alone caused significant hemispheric tissue loss, or lack of growth (2.8 ± 0.42 mm³, p < 0.001). Tissue loss after HI (18.2 ± 5.8 mm³, p < 0.05) was synergistically increased if preceded by IR (32.0 ± 3.5 mm³, p < 0.05). Infarct volume (5.1 ± 1.6 mm³) nearly doubled if HI was preceded by IR (9.8 ± 1.2 mm³, p < 0.05). Pathological scoring revealed that IR aggravated hippocampal, cortical and striatal, but not thalamic, injury. Hippocampal neurogenesis decreased > 50% after IR but was unchanged by HI alone. The number of newly formed microglia was three times higher after IR + HI than after HI alone. In summary, IR to the immature brain produced long‐lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to HI, resulting in larger infarcts, increased hemispheric tissue loss and more inflammation than in non‐irradiated brains.     

Alanine metabolism, transport, and cycling in the brain

Brain glutamate/glutamine cycling is incomplete without return of ammonia to glial cells. Previous studies suggest that alanine is an important carrier for ammonia transfer. In this study, we investigated alanine transport and metabolism in Guinea pig brain cortical tissue slices and prisms, in primary cultures of neurons and astrocytes, and in synaptosomes. Alanine uptake into astrocytes was largely mediated by system L isoform LAT2, whereas alanine uptake into neurons was mediated by Na⁺-dependent transporters with properties similar to system B⁰ isoform B⁰AT2. To investigate the role of alanine transport in metabolism, its uptake was inhibited in cortical tissue slices under depolarizing conditions using the system L transport inhibitors 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid and cycloleucine (1-aminocyclopentanecarboxylic acid; cLeu). The results indicated that alanine cycling occurs subsequent to glutamate/glutamine cycling and that a significant proportion of cycling occurs via amino acid transport system L. Our results show that system L isoform LAT2 is critical for alanine uptake into astrocytes. However, alanine does not provide any significant carbon for energy or neurotransmitter metabolism under the conditions studied.     

Acetate transport and utilization in the rat brain

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-¹³C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR_ace) increased progressively and reached close to saturation for plasma acetate concentration > 2–3 mM, whereas brain acetate concentration continued to increase. The Michaelis–Menten constant for brain acetate utilization (      = 0.01 ± 0.14 mM) was much smaller than for acetate transport through the blood–brain barrier (BBB) (      = 4.18 ± 0.83 mM). The maximum transport capacity of acetate through the BBB (      = 0.96 ± 0.18 μmol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (      = 0.50 ± 0.08 μmol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2–3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.     

Hepatic 31P‐magnetic resonance spectroscopy identified the impact of melatonin‐pretreated mitochondria in acute liver ischaemia‐reperfusion injury

Acute liver ischaemia‐reperfusion injury (IRI), commonly encountered during liver resection and transplantation surgery, is strongly associated with unfavourable clinical outcome. However, a prompt and accurate diagnosis and the treatment of this entity remain formidable challenges. This study tested the hypothesis that ³¹P‐magnetic resonance spectroscopy (³¹P‐MRS) findings could provide reliable living images to accurately identify the degree of acute liver IRI and melatonin‐pretreated mitochondria was an innovative treatment for protecting the liver from IRI in rat. Adult male SD rats were categorized into group 1 (sham‐operated control), group 2 (IRI only) and group 3 (IRI + melatonin [ie mitochondrial donor rat received intraperitoneal administration of melatonin] pretreated mitochondria [10 mg/per rat by portal vein]). By the end of study period at 72 hours, ³¹P‐MRS showed that, as compared with group 1, the hepatic levels of ATP and NADH were significantly lower in group 2 than in groups 1 and 3, and significantly lower in group 3 than in group 1. The liver protein expressions of mitochondrial‐electron‐transport‐chain complexes and mitochondrial integrity exhibited an identical pattern to ³¹P‐MRS finding. The protein expressions of oxidative stress, inflammatory, cellular stress signalling and mitochondrial‐damaged biomarkers displayed an opposite finding of ³¹P‐MRS, whereas the protein expressions of antioxidants were significantly progressively increased from groups 1 to 3. Microscopic findings showed that the fibrotic area/liver injury score and inflammatory and DNA‐damaged biomarkers exhibited an identical pattern of cellular stress signalling. Melatonin‐pretreated mitochondria effectively protected liver against IRI and ³¹P‐MRS was a reliable tool for measuring the mitochondrial/ATP consumption in living animals.     

Decreased TCA cycle rate in the rat brain after acute 3-NP treatment measured by in vivo 1H-[13C] NMR spectroscopy

Inhibition of succinate dehydrogenase (SDH) by the mitochondrial toxin 3-nitropropionic acid (3-NP) has gained acceptance as an animal model of Huntington's disease. In this study 13C NMR spectroscopy was used to measure the tricarboxylic acid (TCA) cycle rate in the rat brain after 3-NP treatment. The time course of both glutamate C4 and C3 13C labelling was monitored in vivo during an infusion of [1-13C]glucose. Data were fitted by a mathematical model to yield the TCA cycle rate (Vtca) and the exchange rate between alpha-ketoglutarate and glutamate (Vx). 3-NP treatment induced a 18% decrease in Vtca from 0.71 +/- 0.02 micro mol/g/min in the control group to 0.58 +/- 0.02 micro mol/g/min in the 3-NP group (p < 0.001). Vx increased from 0.88 +/- 0.08 micro mol/g/min in the control group to 1.33 +/- 0.24 micro mol/g/min in the 3-NP group (p < 0.07). Fitting the C4 glutamate time course alone under the assumption that Vx is much higher than Vtca yielded Vtca=0.43 micro mol/g/min in both groups. These results suggest that both Vtca and Vx are altered during 3-NP treatment, and that both glutamate C4 and C3 labelling time courses are necessary to obtain a reliable measurement of Vtca.     

Glutamate metabolism in cerebral mitochondria after ischemia and post‐ischemic recovery during aging: relationships with brain energy metabolism

Glutamate is involved in cerebral ischemic injury, but its role has not been completely clarified and studies are required to understand how to minimize its detrimental effects, contemporarily boosting the positive ones. In fact, glutamate is not only a neurotransmitter, but primarily a key metabolite for brain bioenergetics. Thus, we investigated the relationships between glutamate and brain energy metabolism in an in vivo model of complete cerebral ischemia of 15 min and during post‐ischemic recovery after 1, 24, 48, 72, and 96 h in 1‐year‐old adult and 2‐year‐old aged rats. The maximum rates (V _max) of glutamate dehydrogenase (GlDH ), glutamate‐oxaloacetate transaminase, and glutamate‐pyruvate transaminase were assayed in somatic mitochondria (FM ) and in intra‐synaptic ‘Light’ mitochondria and intra‐synaptic ‘Heavy’ mitochondria ones purified from cerebral cortex, distinguishing post‐ and pre‐synaptic compartments. During ischemia, none of the enzymes were modified in adult animals. In aged ones, glutamate‐oxaloacetate transaminase was increased in FM and GlDH in intra‐synaptic ‘Heavy’ mitochondria, stimulating glutamate catabolism. During post‐ischemic recovery, FM did not show modifications at both ages while, in intra‐synaptic mitochondria of adult animals, glutamate catabolism was increased after 1 h of recirculation and decreased after 48 and 72 h, whereas it remained decreased up to 96 h in aged rats. These results, with those previously published about Krebs’ cycle and Electron Transport Chain (Villa et al ., [2013] Neurochem. Int . 63, 765–781), demonstrate that: (i) V _max of energy‐linked enzymes are different in the various cerebral mitochondria, which (ii) respond differently to ischemia and post‐ischemic recovery, also (iii) with respect to aging.

     

产前应激对子代大鼠脑缺血/再灌注后星形胶质细胞的影响

目的：探讨产前应激对雄性子代大鼠大脑中动脉缺血/再灌注后星形胶质细胞的影响。方法：SD孕鼠随机分为有产前应激处理（妊娠第15到21天每日3次限制活动）和无产前应激处理,并对其雄性子代大鼠采用线栓法制备大脑中动脉闭塞（MCAO）模型,共分为产前应激+假手术组、MCAO模型组、产前应激+MCAO组（n=10）,于再灌注后第5天检测脑梗死体积,免疫荧光双标染色检测缺血灶边缘区星形胶质细胞形态及促红细胞生成素肝细胞受体A4（EphA4）和胶质纤维酸性蛋白（GFAP）的共表达情况,并采用Western blot检测EphA4、GFAP和神经蛋白聚糖（Neurocan）蛋白表达。结果：产前应激+MCAO组子代大鼠脑梗死体积百分比、EphA4、GFAP和Neurocan蛋白表达均较MCAO组显著增加（P均<0.05）,且GFAP阳性细胞形态学改变及EphA4/GFAP共表达也较MCAO组明显。结论：产前应激可能改变子代大鼠脑缺血/再灌注后星形胶质细胞上EphA4受体的表达,促进星形胶质细胞活化,产生神经蛋白聚糖。     

Dexmedetomidine attenuates neuronal injury after spinal cord ischaemia‐reperfusion injury by targeting the CNPY2‐endoplasmic reticulum stress signalling

Dexmedetomidine (Dex) has been proven to exert protective effects on multiple organs in response to ischaemia‐reperfusion injury, but the specific mechanism by which this occurs has not been fully elucidated. The purpose of this study was to investigate whether Dex attenuates spinal cord ischaemia‐reperfusion injury (SCIRI) by inhibiting endoplasmic reticulum stress (ERS). Our team established a model of SCIRI and utilized the endoplasmic reticulum agonist thapsigargin. Dex (25 g/kg) was intraperitoneally injected 30 minutes before spinal cord ischaemia. After 45 minutes of ischaemia, the spinal cord was reperfused for 24 hours. To evaluate the neuroprotective effect of Dex on SCIRI, neurological function scores were assessed in rats and apoptosis of spinal cord cells was determined by TUNEL staining. To determine whether the endoplasmic reticulum apoptosis pathway CNPY2‐PERK was involved in the neuroprotective mechanism of Dex, the expression levels of related proteins (CNPY2, GRP78, PERK, CHOP, caspase‐12, caspase‐9 and caspase‐3) were detected by western blot analysis and RT‐PCR. We observed that Dex significantly increased the neurological function scores after SCIRI and decreased apoptosis of spinal cord cells. The expression of ERS‐related apoptosis proteins was significantly increased by SCIRI but was significantly decreased in response to Dex administration. Taken together, the results of this study indicate that Dex may attenuate SCIRI by inhibiting the CNPY2‐ERS apoptotic pathway.     

Metabolic flux analysis of CHO cell metabolism in the late non‐growth phase

Chinese hamster ovary (CHO) cell cultures are commonly used for production of recombinant human therapeutic proteins. Often the goal of such a process is to separate the growth phase of the cells, from the non‐growth phase where ideally the cells are diverting resources to produce the protein of interest. Characterizing the way that the cells use nutrients in terms of metabolic fluxes as a function of culture conditions can provide a deeper understanding of the cell biology offering guidance for process improvements. To evaluate the fluxes, metabolic flux analysis of the CHO cell culture in the non‐growth phase was performed by a combination of steady‐state isotopomer balancing and stoichiometric modeling. Analysis of the glycolytic pathway and pentose phosphate pathway (PPP) indicated that almost all of the consumed glucose is diverted towards PPP with a high NADPH production; with even recycle from PPP to G6P in some cases. Almost all of the pyruvate produced from glycolysis entered the TCA cycle with little or no lactate production. Comparison of the non‐growth phase against previously reported fluxes from growth phase cultures indicated marked differences in the fluxes, in terms of the split between glycolysis and PPP, and also around the pyruvate node. Possible reasons for the high NADPH production are also discussed. Evaluation of the fluxes indicated that the medium strength, carbon dioxide level, and temperature with dissolved oxygen have statistically significant impacts on different nodes of the flux network. Biotechnol. Bioeng. 2011; 108:82–92. © 2010 Wiley Periodicals, Inc.     

Synthesis and odor of chiral partial structures of β‐vetivone. I.

Several, stereoisomeric, monocyclic analogs of (−)‐β‐vetivone (1), one of the main constituents of vetiver oil, were studied to determine whether the olfactory properties of (−)‐β‐vetivone (1) could be reproduced from these structurally simpler, synthetically accessible compounds. The effects of diastereomeric and enantiomeric structural differences on the odor of the partial vetivone structure were determined. A chiral phenyl sulfoximine was used for separation of the racemic mixtures. Detailed nuclear magnetic resonance (NMR) spectroscopic studies (¹H, ¹³C) were used to determine relative configurations while absolute configurations were determined by circular dichroism (CD) methods. Chirality 11:14–20, 1999. © 1999 Wiley‐Liss, Inc.     

Synthesis and odor of chiral partial structures of β‐vetivone,Part 2

Several stereoisomeric, monocyclic analogs of (−)‐β‐vetivone (1), one of the main constituents of vetiver oil, were studied to examine if the olfactory properties of (−)‐β‐vetivone (1) could be reproduced from these structurally simpler, synthetically accessible compounds. The effects of diastereomeric and enantiomeric structural differences on the odor of the partial vetivone structure were studied. A chiral phenyl sulfoximine was used for separation of the racemic mixtures. Detailed nuclear magnetic resonance (NMR)‐spectroscopic studies (¹H, ¹³C) were used to determine relative configurations whereas absolute configurations were determined by circular dichroism (CD) methods. Chirality 11:133–138, 1999. © 1999 Wiley‐Liss, Inc.