Direct Detection of Ascorbyl Radical in Experimental Brain Injury: Microdialysis and an Electron Spin Resonance Spectroscopic Study

Abstract: To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H₂O₂ and FeCl₂ adjacent to the dialysis probe. When the rats were challenged with cold-induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl₂ dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.     

Application of in vivo microdialysis to the study of cholinergic systems

The application of in vivo microdialysis to the study of acetylcholine (ACh) release has contributed greatly to our understanding of cholinergic brain systems. This article reviews standard experimental procedures for dialysis probe selection and implantation, perfusion parameters, neurochemical detection, and data analysis as they relate to microdialysis assessments of cholinergic function. Particular attention is focused on the unique methodological considerations that arise when in vivo microdialysis is dedicated expressly to the recovery and measurement of ACh as opposed to other neurotransmitters. Limitations of the microdialysis technique are discussed, as well as methodological adaptations that may prove useful in overcoming these limitations. This is followed by an overview of recent studies in which the application of in vivo microdialysis has been used to characterize the basic pharmacology and physiology of cholinergic neurons. Finally, the usefulness of the microdialysis approach for testing hypotheses regarding the cholinergic systems' involvement in cognitive processes is examined. It can be concluded that, in addition to being a versatile and practical method for studying the neurochemistry of cholinergic brain systems, in vivo microdialysis represents a valuable tool in our efforts to better comprehend ACh's underlying role in a variety of behavioral processes.     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的:应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法:选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果:中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义(P<0.05),乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义(P<0.05),葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义(P<0.05);重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值(P<0.05),乳酸值亦明显高于中度昏迷组测量值(P<0.05),葡萄糖测量值两组术前测量值差异无统计学意义(P>0.05),自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论:结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的：应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法：选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果：中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）,乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义（P〈0．05）,葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）;重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值（P〈0．05）,乳酸值亦明显高于中度昏迷组测量值（P〈O．05）,葡萄糖测量值两组术前测量值差异无统计学意义（P〉0．05）,自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论：结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

Assessment of α-synuclein secretion in mouse and human brain parenchyma

Genetic, biochemical, and animal model studies strongly suggest a central role for α-synuclein in the pathogenesis of Parkinson's disease. α-synuclein lacks a signal peptide sequence and has thus been considered a cytosolic protein. Recent data has suggested that the protein may be released from cells via a non-classical secretory pathway and may therefore exert paracrine effects in the extracellular environment. However, proof that α-synuclein is actually secreted into the brain extracellular space in vivo has not been obtained. We developed a novel highly sensitive ELISA in conjugation with an in vivo microdialysis technique to measure α-synuclein in brain interstitial fluid. We show for the first time that α-synuclein is readily detected in the interstitial fluid of both α-synuclein transgenic mice and human patients with traumatic brain injury. Our data suggest that α-synuclein is physiologically secreted by neurons in vivo. This interstitial fluid pool of the protein may have a role in the propagation of synuclein pathology and progression of Parkinson's disease.     

重型颅脑损伤后颅内高压的治疗研究进展

重型颅脑损伤后颅内压增高预示着不良的神经功能预后和极高的死亡率,一直是临床治疗中的研究热点,可采取高渗性脱水,亚低温疗法,巴比妥昏迷治疗及外科手术干预等治疗措施控制颅内压。由于亚低温治疗会增加患者发生肺炎的风险,巴比妥类药物副作用较大,现均已少用。近来研究发现,监测颅内压、脑灌注压、脑组织氧分压并指导临床治疗,可降低死亡率与改善预后。也有研究发现去骨瓣减压术治疗顽固性颅内高压与神经功能预后较差有关。目前关于颅内高压治疗的最佳方案仍存在争议,未来还需根据患者病情,为其制定规范化与个体化的治疗方案,预防继发性颅脑损伤,降低颅内压。本文就近年来重型颅脑损伤后颅内高压的治疗进展进行阐述。     

血清硫氧还蛋白1与脑外伤患者的白质恢复的相关性分析

为探讨血清硫氧还蛋白1 (thioredoxin 1, Trx1)含量与脑外伤后白质恢复的相关性关系,本研究选取60例脑外伤患者,根据格拉斯哥昏迷指数(Glasgow coma scale, GCS)将患者分为3组(轻度,中度和重度脑外伤患者),每组20例,采用核磁共振成像(magnetic resonance imaging, MRI)检测各组患者头颅白质情况,并通过ELISA方法检测各组患者血清Trx1水平,最后利用SPSS软件Pearson方法检测血清硫氧还蛋白1含量与脑外伤后白质恢复的相关性。结果显示,轻度脑损伤患者的GCS分值明显高于中度和重度脑外伤患者;轻度脑外伤患者的Trx1含量明显高于中度和重度患者;轻度和中度脑外伤患者的白质恢复情况明显优于重度患者;脑损伤患者的血清Trx1含量和白质恢复程度呈正相关。本研究初步结论表明脑外伤患者的白质恢复情况与血清Trx1存在正相关性关系,这将为脑外伤的治疗提供新思路。     

Utilization of in vivo ultrafiltration in biomedical research and clinical applications

Ultrafiltration (UF) is a filtrate selection method with a wide range of biomedical and clinical applications, including detoxification of blood in hemodialysis and peritoneal dialysis. New is, however, the use of UF as a convenient in vivo sampling method that, for example, has been used in diabetics. Ultrafiltration avoids complicated and time-consuming recovery calculations that are necessary when using in vivo microdialysis, as recoveries of low molecular weight molecules are near 100%. The subcutaneously or intravenously placed UF probes have been studied for off-line sample analysis and for continuous on-line monitoring, in a wide variety of species, including dogs, rats, pigs and humans. This review discusses the potential of in vivo UF as a continuous tissue sampling technique in clinical research areas, and in several major biomedical applications including glucose and lactate monitoring and drug kinetic studies.     

In vivo microdialysis in an animal model of neurological disease: thiamine deficiency (Wernicke) encephalopathy

In vivo microdialysis allows for the constant monitoring of brain neurotransmitters in the extracellular fluid of awake and freely moving animals. Considerations including factors affecting probe recoveries, the blood-brain barrier, and tissue reactions to probe implantation are discussed in this paper. Details of the application of in vivo microdialysis to an animal model of encephalopathy are then presented. Thiamine deficiency encephalopathy is an animal model of Wernicke encephalopathy, a neurological disorder observed in alcoholics and in patients with severely compromised nutrition. Regionally selective neuronal cell death is observed in both patients and animals with thiamine deficiency (TD). Various thalamic nuclei suffer significant TD-induced cell death, and NMDA receptor-mediated glutamate excitotoxicity has been proposed as an underlying causative factor. A detailed methodology for the examination of the role of glutamate excitotoxicity using in vivo microdialysis in the neuronal cell death due to thiamine deficiency is presented.     

微透析校正的相关问题和方法

微透析技术是研究生物动态变化的一种新型的活体生物采样技术,近年来由于实验方法的不断改进,微透析技术已广泛应用于在体的定量研究。在进行生物细胞外液的定量研究中,微透析探针的校正是十分必要的。本从微透析的回收率、影响因素及校正方法等方面简要介绍了微透析校正的相关问题。     

微透析技术在脑靶向中应用

微透析技术作为一门新兴的技术,近年来多用于靶向分布和体内代谢等方面,尤其是在药物的脑部研究方面,该技术显得尤为重要。如今,随着新型探针的不断出现,以及微量、快速、灵敏的分析检测手段的发展,微透析技术已日益成为药物脑部研究的重要工具。现通过检索近十年来的相关文献,对脑微透析技术的概况、原理、脑微透析探针以及其应用作一综述,希望能为从事该方面研究的药学工作者提供相关参考。     

脑内微透析采样技术及其在神经科学中的应用

作为一种新的在体化学采样技术,脑内微透析引起了神经科学家的关注。它与迅速发展起来的高灵敏度的微量分析技术相结合,实现了对体内细胞外环境中化学物质变化的动态监测,从而在神经科学领域获得应用。本系统地介绍了这一新技术的原理和方法,并扼要地介绍了一这一技术在神经科学中的应用及其取得的新进展,并结合本实验室的工作经验,对该技术存在的一些问题进行了讨论。     

In vivo monitoring of cerebral agmatine by microdialysis and capillary electrophoresis

Agmatine is a putative neurotransmitter in the brain. Current analytical techniques do not allow the detection of agmatine in extracellular fluid, making it difficult to study its physiological role. However, a new method for in vivo monitoring agmatine in the brain was developed. Capillary zone electrophoresis and laser induced fluorescence detection (CZE-LIFD) was used to measure nanomolar concentrations of agmatine in submicroliter sample volumes. This analytical technique proved to detect 0.49 attomole of agmatine improving the sensitivity of previous analytical techniques. On the other hand, the hippocampus is a brain region well known for having a population of agmatine containing neurons. Therefore, intracerebral microdialysis was performed in the hippocampus and agmatine was extracted from the extracellular environment. Detectable amounts of agmatine were found in dialysates from probes located in the hippocampus but not from the probes located in the lateral ventricle. Furthermore, extracellular agmatine was calcium and impulse dependent and depolarization of hippocampal neurons increased extracellular agmatine concentration. The methods reported here are sensitive enough to study the physiological role of brain agmatine in freely moving animals.     

Model for Concomitant Microdialysis Sampling of the Pons and Cerebral Cortex in Rhesus Macaques (Macaca mulatta)

Pediatric diffuse intrinsic pontine gliomas are aggressive brainstem tumors that fail to respond to treatment. We hypothesize that the protective features of the pons may hinder chemotherapeutic agents from entering pontine tissue compared with cortical brain tissue. To test this hypothesis, we developed a unique nonhuman primate model using microdialysis, a continuous in vivo extracellular sampling technique, to compare drug exposure concurrently in pontine tissue, cortical tissue, CSF, and plasma after intravenous administration of chemotherapeutic agents. The surgical coordinates and approach for microdialysis cannula–probe placement were determined in 5 adult male rhesus monkeys (Macaca mulatta) by using MRI. Microdialysis cannulas–probes were implanted stereotactically in the brain, retrodialysis was performed to measure relative recovery, and a 1-h intravenous infusion of temozolomide was administered. Continuous microdialysis samples were collected from the pons and cortex over 4 h with concurrent serial plasma and CSF samples. Postsurgical verification of microdialysis cannula–probe placement was obtained via MRI in 3 macaques and by gross pathology in all 5 animals. The MRI-determined coordinates and surgical methodologies resulted in accurate microdialysis probe placement in the pons and cortex in 4 of the 5 macaques. Histologic examination from these 4 animals revealed negligible tissue damage to the pontine and cortical tissue from microdialysis. One macaque was maintained for 8 wk and had no deficits attributed to the procedure. This animal model allows for the determination of differences in CNS penetration of chemotherapeutic agents in the pons, cortex, and CSF after systemic drug administration.Abbreviations: DIPG, diffuse intrinsic pontine glioma; ECF, extracellular fluidPediatric diffuse intrinsic pontine gliomas (DIPG) are aggressive tumors that cannot be surgically resected due to their location, and are resistant to chemotherapeutic and radiation therapies. As a result, children with DIPG have a dismal prognosis with median survival less than one year from diagnosis. One hypothesis for the poor efficacy of treatment is that innate CNS protective features, such as the blood–brain barrier and the blood–CSF barrier, shield the brainstem to a higher degree given its critical functions, and isolate pontine gliomas from treatment. To test this hypothesis, we developed a nonhuman primate model in rhesus monkeys to evaluate pontine tissue pharmacokinetics by using microdialysis, a continuous in vivo extracellular sampling technique based on diffusion. Microdialysis is the ‘gold standard’ for in vivo sampling methodologies in the CNS, enabling the collection of extracellular tissue fluid via passive diffusion by using a semipermeable membrane probe.A nonhuman primate model demonstrating the feasibility of microdialysis sampling from cortical brain tissue with concurrent pharmacokinetic sampling during chemotherapeutic drug administration has previously been established,^3-5,7 but there are no current animal models that measure drug penetration into the pons. The location of the pons deep within the brain, as well as the vital brainstem functions associated with the pons, present additional obstacles to accurate microdialysis probe placement and sample collection. The objectives of the current study were to develop imaging and surgical procedures for the accurate placement of a microdialysis probe within the pons of rhesus monkeys for sample collection, to establish a method to perform microdialysis simultaneously in multiple CNS regions, and to develop a mechanism to perform repeated microdialysis in the same areas with a single invasive surgical procedure. This model allows for the pharmacokinetic comparison of drug penetration into pontine tissue, in conjunction with cortical tissue, plasma, and CSF, after intravenous administration.     

Lactography as an approach to monitor glucose metabolism on-line in brain and muscle

1. Thus far metabolic processes in the intact animal (or man) have been studied either by the analysis of body fluids, of biopsies, of tissue obtained post mortem or by techniques, requiring dedicated and expensive equipment (such as positron emission tomography or magnetic resonance spectroscopy). 2. Here we describe a relatively simple and inexpensive technique, that can be applied in vivo to study metabolism in brain regions and muscle in the freely moving rat and in human peripheral tissue. 3. The method is based on microdialysis allowing continuous sampling from the extracellular space, the enzymatic conversion of lactate and the on-line detection of fluorescent NADH. 4. Examples of the application of our technique include the monitoring of lactate efflux from various brain regions of behaving animals under a variety of stress exposures, during ischemia or hypoxia and drug treatments. 5. The results indicate that in brain lactate is not exclusively formed under hypoxia and that neuronal activation leads also to lactate formation, possibly due to the compartmentation of both the involved enzymes and the energy metabolism. 6. The increase of lactate formation in contracting or ischemic muscle or during exercise could also be followed on-line in the rat, suggesting that our approach allows the continuous monitoring of anaerobic metabolism in man e.g. during traumatic or arteriosclerotic limb ischemia or lactic acidosis in shock states. 7. The principle of our approach can easily be adapted to other metabolites, thus enabling to monitor other metabolic pathways in vivo as well.     

The brain in vivo expresses the 2',3'-cAMP-adenosine pathway

Although multiple biochemical pathways produce adenosine, studies suggest that the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) contributes to adenosine production in some cells/tissues/organs. To determine whether the 2',3'-cAMP-adenosine pathway exists in vivo in the brain, we delivered to the brain (gray matter and white matter separately) via the inflow perfusate of a microdialysis probe either 2',3'-cAMP, 3',5'-cAMP, 2'-AMP, 3'-AMP, or 5'-AMP and measured the recovered metabolites in the microdialysis outflow perfusate with mass spectrometry. In both gray and white matter, 2',3'-cAMP increased 2'-AMP, 3'-AMP and adenosine, and 3',5'-cAMP increased 5'-AMP and adenosine. In both brain regions, 2'-AMP, 3-AMP and 5'-AMP were converted to adenosine. Microdialysis experiments in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) wild-type mice demonstrated that traumatic brain injury (controlled cortical impact model) activated the brain 2',3'-cAMP-adenosine pathway; similar experiments in CNPase knockout mice indicated that CNPase was involved in the metabolism of endogenous 2',3'-cAMP to 2'-AMP and to adenosine. In CSF from traumatic brain injury patients, 2',3'-cAMP was significantly increased in the initial 12 h after injury and strongly correlated with CSF levels of 2'-AMP, 3'-AMP, adenosine and inosine. We conclude that in vivo, 2',3'-cAMP is converted to 2'-AMP/3'-AMP, and these AMPs are metabolized to adenosine. This pathway exists endogenously in both mice and humans.     

Triggers and mediators of hemorrhagic transformation in cerebral ischemia

Intracerebral hemorrhagic transformation is a multifactorial phenomenon in which ischemic brain tissue converts into a hemorrhagic lesion with blood-vessel leakage, extravasation, and further brain injury. It has been estimated that up to 30-40% of all ischemic strokes undergo spontaneous hemorrhagic transformation, and this phenomenon may become even more prevalent with the increasing use of thrombolytic stroke therapy. An emerging conceptual model suggests that the loss of microvascular integrity and disruption of neurovascular homeostasis connects the experimental findings of blood-cell extravasation to brain injury after hemorrhage. In this short article, we examine mechanisms related to reperfusion injury and oxidative stress, leukocyte infiltration, vascular activation, and dysregulated extracellular proteolysis as potential triggers of hemorrhagic transformation. Perturbations in cell-cell and cell-matrix signaling within the hypothesized neurovascular unit may ultimately lead to neuroinflammation and apoptotic-like cell death in the parenchyma. Further investigations into the molecular mediators of hemorrhagic transformation may reveal new therapeutic targets for this clinically complex problem.     

Lactate and the Lactate-to-Pyruvate Molar Ratio Cannot Be Used as Independent Biomarkers for Monitoring Brain Energetic Metabolism: A Microdialysis Study in Patients with Traumatic Brain Injuries

Background

For decades, lactate has been considered an excellent biomarker for oxygen limitation and therefore of organ ischemia. The aim of the present study was to evaluate the frequency of increased brain lactate levels and the LP ratio (LPR) in a cohort of patients with severe or moderate traumatic brain injury (TBI) subjected to brain microdialysis monitoring to analyze the agreement between these two biomarkers and to indicate brain energy metabolism dysfunction.

Methods

Forty-six patients with an admission Glasgow coma scale score of ≤13 after resuscitation admitted to a dedicated 10-bed Neurotraumatology Intensive Care Unit were included, and 5305 verified samples of good microdialysis data were analyzed.

Results

Lactate levels were above 2.5 mmol/L in 56.9% of the samples. The relationships between lactate and the LPR could not be adequately modeled by any linear or non-linear model. Neither Cohen’s kappa nor Gwet’s statistic showed an acceptable agreement between both biomarkers to classify the samples in regard to normal or abnormal metabolism. The dataset was divided into four patterns defined by the lactate concentrations and the LPR. A potential interpretation for these patterns is suggested and discussed. Pattern 4 (low pyruvate levels) was found in 10.7% of the samples and was characterized by a significantly low concentration of brain glucose compared with the other groups.

Conclusions

Our study shows that metabolic abnormalities are frequent in the macroscopically normal brain in patients with traumatic brain injuries and a very poor agreement between lactate and the LPR when classifying metabolism. The concentration of lactate in the dialysates must be interpreted while taking into consideration the LPR to distinguish between anaerobic metabolism and aerobic hyperglycolysis.     

Extracellular glutamate changes in rat striatum during ischemia determined by a novel dialysis electrode and conventional microdialysis

Our newly developed method using a dialysis electrode has made it possible to perform real time monitoring of extracellular glutamate concentration ([Glu]e) utilizing the oxygen-independent reaction with glutamate oxidase and ferrocene. In this study, we therefore, investigated [Glu]e changes during brain ischemia using both the conventional microdialysis method and the dialysis electrode method. A comparison between our newly developed dialysis electrode and conventional microdialysis methods provided the following results. When the conventional microdialysis method was employed: (1) the elevation of [Glu]e during complete global ischemia was delayed; and (2) the elevation of concentration and reuptake of glutamate were delayed during 10-min transient ischemia, and the elevation of [Glu]e reached a maximum later using conventional microdialysis than using our dialysis electrode. (3) The biphasic [Glu]e elevation of glutamate concentration detected using the dialysis electrode method was not observed using the conventional microdialysis method. It was additionally investigated why the conventional microdialysis method provides inferior time resolution. In this study, we also demonstrated with the chromatographic SMART procedure coupled to UV detection that biogenic substances, i.e. low molecular weight proteins and peptides, are released during ischemic injury, and they may cause a delay in the time resolution in the microdialysis method.     

Induction of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-9 via CD47 Signaling in Neurovascular Cells

Neurovascular injury comprises a wide spectrum of pathophysiology that underlies the progression of brain injury after cerebral ischemia. Recently, it has been shown that activation of the integrin-associated protein CD47 mediates the development of blood–brain barrier injury and edema after cerebral ischemia. However, the mechanisms that mediate these complex neurovascular effects of CD47 remain to be elucidated. Here, we compare the effects of CD47 signaling in brain endothelial cells, astrocytes, and pericytes. Exposure to 4N1 K, a specific CD47-activating peptide derived from the major CD47 ligand thrombospondin-1, upregulated two major neurovascular mediators, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9), in brain endothelial cells and astrocytes. No changes were detected in pericytes. These findings may provide a potential mechanism for CD47-induced changes in blood–brain barrier homeostasis, and further suggest that CD47 may be a relevant neurovascular target in stroke.