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Simultaneous detection of mitochondrial respiratory chain activity and reactive oxygen in digitonin-permeabilized cells using flow cytometry.

BACKGROUND: Increased mitochondrial generation of reactive oxygen intermediates (ROI) due to defective respiratory chain activity has been implicated in physiological processes such as apoptosis, in the pathogenesis of mitochondrial diseases, and as part of the normal aging process. Established methods addressing activity of the respiratory chain complexes have been limited to bulk assays for single parameters. This study describes a flow cytometry-based method and its validation for the detection of respiratory chain function in single cells permeabilized by digitonin. METHODS: Flow cytometry was used to measure mitochondrial membrane potential (DeltaPsi(m)) and reactive oxygen generation under differing conditions of respiration. This was brought about by the addition of substrates and inhibitors to digitonin-permeabilized cells. This method was validated by measurement of oxygen consumption and ATP production and by confocal microscopy. RESULTS: Activity of the respiratory chain complexes assessed by DeltaPsi(m) responded to substrates and inhibitors as predicted from assessment by oxygen consumption and ATP synthesis. In addition, the flow cytometry method allows the simultaneous assessment of mitochondrial ROI generation. This was confirmed by the localization of the ROI probe, carboxy-DCF, to the same site as the mitochondrial probe observed by confocal microscopy. CONCLUSIONS: This method allows the functional integrity of the respiratory chain complexes to be studied at the single-cell level, thus addressing the relationship between disordered function of respiratory chain complexes and mitochondrial ROI generation.

Adenosine Triphosphate↗

High-resolution random mesh algorithms for creating a probabilistic 3D surface atlas of the human brain.

Striking variations exist, across individuals, in the internal and external geometry of the brain. Such normal variations in the size, orientation, topology, and geometric complexity of cortical and subcortical structures have complicated the problem of quantifying deviations from normal anatomy and of developing standardized neuroanatomical atlases. This paper describes the design, implementation, and results of a technique for creating a three-dimensional (3D) probabilistic surface atlas of the human brain. We have developed, implemented, and tested a new 3D statistical method for assessing structural variations in a data-base of anatomic images. The algorithm enables the internal surface anatomy of new subjects to be analyzed at an extremely local level. The goal was to quantify subtle and distributed patterns of deviation from normal anatomy by automatically generating detailed probability maps of the anatomy of new subjects. Connected systems of parametric meshes were used to model the internal course of the following structures in both hemispheres: the parieto-occipital sulcus, the anterior and posterior rami of the calcarine sulcus, the cingulate and marginal sulci, and the supracallosal sulcus. These sulci penetrate sufficiently deeply into the brain to introduce an obvious topological decomposition of its volume architecture. A family of surface maps was constructed, encoding statistical properties of local anatomical variation within individual sulci. A probability space of random transformations, based on the theory of Gaussian random fields, was developed to reflect the observed variability in stereotaxic space of the connected system of anatomic surfaces. A complete system of probability density functions was computed, yielding confidence limits on surface variation. The ultimate goal of brain mapping is to provide a framework for integrating functional and anatomical data across many subjects and modalities. This task requires precise quantitative knowledge of the variations in geometry and location of intracerebral structures and critical functional interfaces. The surface mapping and probabilistic techniques presented here provide a basis for the generation of anatomical templates and expert diagnostic systems which retain quantitative information on intersubject variations in brain architecture.

Algorithms↗

Endothelial function. From vascular biology to clinical applications.

The endothelium, by releasing nitric oxide (NO), promotes vasodilation and inhibits inflammation, thrombosis, and vascular smooth muscle cell proliferation. These biological actions of NO make it an important component in the endogenous defense against atherosclerosis and its overt clinical complications. Loss of the functional integrity of the endothelium, as seen commonly in the milieu of cardiovascular risk factors, plays an integral role in all stages of atherosclerosis from lesion initiation to plaque rupture. A number of established techniques can assess endothelial function in human vascular beds. The outcome of endothelial testing has profound prognostic implications and is an independent predictor of atherosclerosis disease progression and cardiovascular event rates. The large clinical benefit of statins and angiotensin-converting enzyme inhibitors in patients with atherosclerosis involves favorable effects of endothelial function. Studies of endothelial function represent a prime example of a successful application of insights derived from vascular biology at the bedside.

Coronary Artery Disease↗

The anatomical relationship of the prefrontal cortex with the striatopallidal system, the thalamus and the amygdala: evidence for a parallel organization.

Recent findings in primates indicate that the connections of the frontal lobe, the basal ganglia, and the thalamus are organized in a number of parallel, functionally segregated circuits. In the present account, we have focused on the organization of the connections between the prefrontal cortex, the basal ganglia and the mediodorsal thalamic nucleus in the rat. It is concluded that in this species, in analogy with the situation in primates, a number of parallel basal ganglia-thalamocortical circuits exist. Furthermore, data are presented indicating that the projections from particular parts of the amygdala and from individual nuclei of the midline and intralaminar thalamic complex to the prefrontal cortex and the striatum are in register with the arrangements in the parallel circuits. These findings emphasize that the functions of the different subregions of the prefrontal cortex cannot be considered separately but must be viewed as components of the integrative functions of the circuits in which they are involved.

Amygdala↗

The hippocampus: functional and structural correlations.

This paper reviews the current literature relating neuropsychological measures of the functional integrity of the hippocampus with structural indices obtained from magnetic resonance imaging (MRI) volumetric scans and the histological analysis of postoperative specimens. The first part of the discussion presents an overview of the current methods and applications of volumetric MRI of the hippocampus. The processes and contemporary standards applied to the histological analysis of resected hippocampal tissue are also described. Both of these measures of structural integrity have been studied in relation to preoperative performance on neuropsychological tests, including the intracarotid sodium amytal test, and postoperative memory functioning. The findings from these studies are reviewed. The challenges presented by these new technologies to the field of neuropsychology are discussed.

Brain Mapping↗

Integration of single cell injection, cell lysis, separation and detection of intracellular constituents on a microfluidic chip.

A microfluidic system was developed for the analysis of single biological cells, with functional integration of cell sampling, single cell loading, docking, lysing, and capillary electrophoretic (CE) separation with laser induced fluorescence (LIF) detection in microfabricated channels of a single glass chip. Channels were 12 microm deep and 48 microm wide, with a simple crossed-channel design. The effective separation channel length was 35 mm. During sampling with a cell suspension (cell population 1.2 x 10(5) cells per mL in physiological salt solution), differential hydrostatic pressure (created by adjusting liquid levels in the four reservoirs) was used to control cell flow exclusively through the channel crossing. Single cell loading into the separation channel was achieved by electrophoretic means by applying a set of potentials at the four reservoirs, counteracting the hydrostatic flow. A special docking (adhering) procedure for the loaded cell was applied before lysis by repeatedly connecting and disconnecting a set of low potentials, allowing precise positioning of the cell within the separation channel. Cell lysis was then effected within 40 ms under an applied CE separation voltage of 1.4 kV (280 V cm(-1)) within the working electrolyte (pH 9.2 borate buffer) without additional lysates. The docked lysing approach reduced dispersion of released intracellular constituents, and significantly improved the reproducibility of CE separations. Glutathione (GSH) was used as a model intracellular component in single human erythrocyte cells. NDA derivatized GSH was detected using LIF. A throughput of 15 samples h(-1), a retention time precision of 2.4% RSD was obtained for 14 consecutively injected cells. The average cellular concentration of GSH in human erythrocytes was found to be 7.2 [times] 10(-4)+/- 3.3 x 10(-4) M (63 +/- 29 amol per cell). The average separation efficiency for GSH in lysed cells was 2.13 x 10(6)+/- 0.4 x 10(6) plates per m, and was about a factor of 5 higher than those obtained with GSH standards using pinched injection.

Electrophoresis, Capillary↗

Pursuit eye movement deficits in autism.

Oculomotor studies provide a novel strategy for evaluating the functional integrity of multiple brain systems and cognitive processes in autism. The current study compared pursuit eye movements of 60 high-functioning individuals with autism and 94 intelligence quotient, age and gender matched healthy individuals using ramp and oscillating target tasks. Individuals with autism had normal pursuit latency, but reduced closed-loop pursuit gain when tracking both oscillating and ramp targets. This closed-loop deficit was similar for leftward and rightward pursuit, but the difference between individuals with autism and their age-matched peers was more apparent after mid-adolescence, suggesting reduced maturational achievement of the pursuit system in autism. Individuals with autism also had lower open-loop pursuit gain (initial 100 ms of pursuit) and less accurate initial catch-up saccades during a foveofugal step-ramp task, but these deficits were only seen when targets moved into the right visual field. Pursuit performance in both open- and closed-loop phases was correlated with manual praxis in individuals with autism. Bilateral disturbances in the ability to use internally generated extraretinal signals for closed-loop pursuit implicate frontostriatal or cerebellar circuitry. The hemifield specific deficit in open-loop pursuit demonstrates a lateralized disturbance in the left extrastriate areas that extract visual motion information, or in the transfer of visual motion information to the sensorimotor areas that transform visual information into appropriate oculomotor commands.

Adolescent↗

LDH-C4 in human seminal plasma and testicular function. III. Relationship to other semen variables.

Lactic dehydrogenase C4 was analysed in human seminal plasma, expressed in nanokatals and related to the sperm count. The LDH-C4/sperm ratio has previously been shown to reflect the functional integrity of the seminiferous epithelium. In the present study this ratio was related to several other semen variables that reflect various functional or biochemical properties of the spermatozoa or other cell systems. For none of these variables was there a significant linear correlation with the LDH-C4/sperm ratio, but interesting differences became apparent when the semen samples were subdivided into two groups, those with normal (less than 20) and those with a high (greater than 20) ratio. For nuclear chromatin stability, oxygen consumption, succinate-induced increase in oxygen consumption and lipid peroxidation, there were significant differences between the two groups. There was no difference between the groups for the staining properties of the DNA-DNP complex, zinc, fructose and free 1-carnitine content in the ejaculate, and the zinc/fructose ratio. The results indicate that functional properties of the spermatozoa can be related to the status of the seminiferous epithelium.

Cell Nucleus↗

Dysautonomia in fatal familial insomnia as an indicator of the potential role of the thalamus in autonomic control.

Fatal familial insomnia (FFI) is characterized by insomnia, dysautonomia, disruption of circadian rhythms, and motor dysfunction. The typical neuropathological findings in FFI are severe neuronal depletion in the mediodorsal (MD) and anteroventral nuclei of the thalamus. The interaction between the thalamus and central autonomic control mechanisms is poorly understood. The central autonomic areas include the anterior cingulate and insular cortices; amygdala, paraventricular nucleus, dorsomedial nucleus, and lateral hypothalamic area; periaqueductal gray; parabrachial nucleus; ventrolateral medulla; and nucleus of the solitary tract. Several nuclei of the thalamus have connections with areas of the central autonomic network. The paraventricular nucleus (PVT) projects to the medial prefrontal cortex, and receives multimodal visceral and somatosensory inputs. The MD nucleus is connected with several "limbic" areas involved in autonomic control. The autonomic manifestations of FFI are exaggerated sympathetic activation with preserved parasympathetic drive to the cardiovascular system. This reflects an exaggerated sympathetic drive from supramedullary structures. Bicuculline, administered into the MD, elicits an increase in arterial pressure and heart rate. The medial portion of the MD may share with the PVT a relay function for circuits controlling autonomic responses. MD involvement in FFI suggests a role of the thalamus in central autonomic and other integrative functions.

Autonomic Nervous System↗

Motion perception in autism: a "complex" issue.

We present the first assessment of motion sensitivity for persons with autism and normal intelligence using motion patterns that require neural processing mechanisms of varying complexity. Compared to matched controls, our results demonstrate that the motion sensitivity of observers with autism is similar to that of nonautistic observers for different types of first-order (luminance-defined) motion stimuli, but significantly decreased for the same types of second-order (texture-defined) stimuli. The latter class of motion stimuli has been demonstrated to require additional neural computation to be processed adequately. This finding may reflect less efficient integrative functioning of the neural mechanisms that mediate visuoperceptual processing in autism. The contribution of this finding with regards to abnormal perceptual integration in autism, its effect on cognitive operations, and possible behavioral implications are discussed.

Adolescent↗

Age and immunity.

Longitudinal studies are defining progressive alterations to the immune system associated with increased mortality in the very elderly. Many of these changes are exacerbated by or even caused by chronic T cell stimulation by persistent antigen, particularly from Cytomegalovirus. The composition of T cell subsets, their functional integrity and representation in the repertoire are all markedly influenced by age and by CMV. How these findings relate to epidemiological, functional, genetic, genomic and proteomic studies of human T cell immunosenescence was the subject of intense debate at an international conference held just before Christmas 2005 in the Black Forest.

Journal Article↗

[Methods to assess the ex vivo growth capacity of mesothelial cells obtained directly from peritoneal effluent].

The anatomical and functional integrity of mesothelial cells (MC) is necessary for peritoneal membrane stability. At present, there is no satisfactory method to assess MC function and regenerative capacity in individual peritoneal dialysis (PD) patients. MC may be cultured from peritoneal biopsy specimens, but peritoneal biopsy is an invasive procedure that cannot be performed serially. The aim of this study is to explore the feasibility of serial culture of MC from the peritoneal effluent of PD patients. Fifty-two randomly selected PD patients were studied. MC were obtained from the peritoneal effluent of nocturnal 2.27% glucose exchanges and cultured in T25 tissue culture flasks. Subconfluent MC cultures were obtained in 80.7% of patients. At this stage, the percentage of cells in the tissue cultured flask characterized as MC by morphology and immunostaining had increased to 95.5%. MC were then subcultured in multi-well culture plates, where they showed exponential cell growth until day 16. Nine (17%) patients released low numbers of MC into the effluent and MC could not be cultured to subconfluence. One additional patient released and apparently adequate number of MC that repeatedly failed to reach confluence. Patients showed the same behavior in several cultures performed. In conclusion, peritoneal MC released into peritoneal effluent are accessible for profound analysis by a culture technique. This technique opens the possibility of serial follow-up of the biology of MC individual PD patients.

Cell Division↗

Improvement of lacrimal function by topical application of CyA in murine models of Sjögren's syndrome.

PURPOSE: The object of this study was to evaluate improvement of lacrimal gland (LG) function after topical cyclosporin A (CyA). METHODS: Topical CyA (0.01% and 0.1%) was applied to two mouse models of Sjögren's syndrome, the NFS/sld after thymectomy and the nonobese diabetic (NOD) mouse, and the functional integrity of the lacrimal gland was evaluated by measuring basal and stimulated tear secretion and its histologic integrity by examining it for apoptosis and lymphocyte invasion. RESULTS: After treatment with CyA at 0.1% in the NFS/sld mice, tear function increased, and there was a decrease in lymphocyte infiltration of the LG and a decrease in apoptotic figures among the acinar cells. In the NOD mice, tear function also improved, but there was no associated decrease in lymphocyte infiltration. However, the expression of Fas ligand (FasL) in NOD mice by infiltrating lymphocytes was suppressed with 0. 1% CyA eye drops. CONCLUSIONS: CyA appears to improve tear secretion in mouse models of Sjögren's syndrome by preventing lymphocyte-induced apoptosis of acinar cells. In one model this was achieved by preventing lymphocyte infiltration and in the other by reducing expression of FasL expression on infiltrating lymphocytes.

Administration, Topical↗

[Do we defend a brain oriented view of death?].

AIMS: To propose a new formulation of death based on the mechanisms involved in consciousness generation in human beings. DEVELOPMENT: Any complete formulation of death must include three different elements: a definition of death, its anatomofunctional substratum and the tests required to diagnose death. The three brain oriented formulations of death are: the whole brain, the brain stem, and the neocortical formulations of death. In this paper I review and discuss each of these formulations, and I propose a new formulation about human death based on the physiopathological mechanisms involved in the generation of consciousness. This contains two physiological components: arousal and awareness. Since the structures of the brain stem, the diencephalon and the cerebral cortex interact to generate consciousness, it would be a mistake to make a rigid distinction between their functions from the point of view of waking and content. Important interconnections between the brain stem, other subcortical structures and the neocortex give rise to both components of consciousness. The generation of consciousness, then, is based on the anatomy and the physiology of pathways throughout the whole brain. None of the three formulations above is wholly satisfactory. CONCLUSION: I propose a new formulation of death that identifies consciousness as the most important function of the organism, because it provides the essential human attributes and the highest level of control within the hierarchy of integrating functions of the organism

Arousal↗

[The effects of long-term sedation on intestinal function].

Gastrointestinal integrity with intact function are of main importance in critically ill patients, and not only as a route of nutritional support. Drugs used for long-term sedation can lead to disordered gastrointestinal motility. In this study we compared the influence of different combinations of analgesics and sedatives on the intestinal function in mechanically ventilated, critically ill patients. METHODS. A total of 190 patients were evaluated retrospectively. All patients required controlled mechanical ventilation and deep sedation (Ramsay Score 5-6) for 7 days or more due to acute respiratory failure or elevated intracranial pressure. In none of these patients was enteric tube feeding contraindicated. Intact intestinal function was assumed when full enteric tube feeding was achieved on days 5 and 6 of the treatment period. Furthermore, other gastrointestinal motility disorders (e.g. constipation) had to be absent. In all patients the feeding tube was placed in the stomach by the nasogastric route. Corresponding to different combinations of analgesics and sedatives, the 190 patients were divided into 11 groups. The following combinations were used: group 1 (n = 20), fentanyl+flunitrazepam; group 2 (n = 20), fentanyl+midazolam; group 3 (n = 20), fentanyl+thiopentone; group 4 (n = 20) piritramide+midazolam; group 5 (n = 20), piritramide and continuous epidural administration of bupivacaine+midazolam; group 6 (n = 20), piritramide+gamma-aminobutyric acid (GABA); group 7 (n = 20), ketamine+midazolam; group 8 (n = 10), ketamin+methohexitone; group 9 (n = 20), ketamine+propofol; group 10 (n = 10), ketamine+midazolam and GABA; group 11 (n = 10), sufentail+midazolam and methohexitone. Patients in groups 3, 8, 9, 10, and 11 all had severe head injury and elevated intracranial pressure. Group 6 was made up exclusively of elderly patients (> 65 years) without head trauma. RESULTS. The patients in groups 1, 2, and 3 received fentanyl for analgesia and were completely fed by enteric tube in 30%, 35%, and 15% of cases, respectively. In group 3 deep sedation was necessary because of elevated intracranial pressure. In groups 4, 5, and 6, piritramide was administered for analgesia, and normal enteric tube feeding was achieved in 70%, 75%, and 90% of cases. The best results were seen in group 6, and these elderly patients needed smaller amounts of piritramide for analgesia. In groups 7, 8, 9, and 10, ketamine was given for analgesia, and complete enteric tube feeding was carried out in 75%, 30%, 45%, and 60% of these patients. The best results in the ketamine groups were found in combination with midazolam as the sedating drug; however, the patients in group 7 did not have elevated intracranial pressure, in contrast to the patients in groups 8, 9, and 10. The last group received the combination of sufentanil, midazolam and methohexitone to achieve a deep sedation. The rate of normal enteric tube feeding in these patients with severe head trauma was 30%. CONCLUSIONS. In patients with severe head trauma who need deep sedation to prevent dangerous high intracranial pressure, gastrointestinal motility disorders are very commonly found. The results obtained suggest that ketamine should be regarded as the analgesic drug of choice, combined with propofol rather than a high-dose barbiturate therapy. The combination of ketamine with midazolam and GABA is an unusual strategy for long-term sedation, which resulted from our own clinical studies directed at an effective and well-tolerated regime for this high-risk patient group. Obviously, high-dose barbiturates and short-acting opioids, especially when combined, make enteric tube feeding more difficult. Therefore, we recommend piritramide or ketamine for analgesia. The basic sedating drug is midazolam, in special cases combined with or replaced by propofol. The position of GABA in long-term sedation is not yet clear, but a lack of side effects on the gastrointestinal tract became evident in this stud

Adolescent↗

Integrative analysis of cell cycle control in budding yeast.

The adaptive responses of a living cell to internal and external signals are controlled by networks of proteins whose interactions are so complex that the functional integration of the network cannot be comprehended by intuitive reasoning alone. Mathematical modeling, based on biochemical rate equations, provides a rigorous and reliable tool for unraveling the complexities of molecular regulatory networks. The budding yeast cell cycle is a challenging test case for this approach, because the control system is known in exquisite detail and its function is constrained by the phenotypic properties of >100 genetically engineered strains. We show that a mathematical model built on a consensus picture of this control system is largely successful in explaining the phenotypes of mutants described so far. A few inconsistencies between the model and experiments indicate aspects of the mechanism that require revision. In addition, the model allows one to frame and critique hypotheses about how the division cycle is regulated in wild-type and mutant cells, to predict the phenotypes of new mutant combinations, and to estimate the effective values of biochemical rate constants that are difficult to measure directly in vivo.

Cell Cycle↗

Dopamine: a key regulator to adapt action, emotion, motivation and cognition.

Dopamine is expressed in restricted brain areas involved in numerous integrative functions contributing to automated behaviours that are highly adaptive. During ontogenesis, dopamine can have a trophic action, which influences cortical specification directly, especially in prefrontal areas. Such a role is attested by the close relationships existing between the development of dopamine cortical innervation and cognitive abilities. Interestingly, such a proposal is reinforced by phylogenetic data. During the last stages of evolution in mammals, the characteristic extension of dopamine cortical innervation is also correlated with the development of cognitive capacities. More generally, the contribution of dopamine will be to increase the processing of cortical information through basal ganglia, either during the course of evolution or development. In this respect, dysmaturation suspected in schizophrenia and attention deficit-hyperactivity disorder in children can emphasize such a defect in basal ganglia processing. Remarkably, these diseases are improved by dopamine antagonists and agonists, respectively. In this line of evidence, experimental studies showed that selective lesions of the dopamine neurones in rats or primates can actually provide motor, limbic and cognitive deficits, in later cases especially when the mesocorticolimbic dopamine pathways are altered. Data resulting from lesion studies also showed significant alteration in attentional processes, thus raising the question of the direct involvement of dopamine in regulating attention. Dopamine can act as a powerful regulator and integrator of different aspects of brain functions. For example, in Parkinson's disease, besides motor impairment, dopamine degeneration is also expressed by alterations of both limbic, executive and cognitive functions, both improved by dopamine receptor agonists and dopa therapy. Dopamine has thus to be considered as a key regulator that contributes to behavioural adaptation and to the anticipatory processes necessary for preparing voluntary action consequent upon intention. In this respect, the alteration of dopamine transmission with age could contribute to cognitive impairment. Therefore, to normalize dopamine transmission pharmacologically could actually improve the cognitive and limbic deficits during normal aging, as it does in psychiatric and neurological disorders.

Animals↗

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip.

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.

Bacteriophage lambda↗