Combined effects of aquaporin-4 and hypoxia produce age-related hydrocephalus

Aquaporin-4, present in ependymal cells, in glia limiting and abundantly in pericapillary astrocyte foot processes, and aquaporin-1, expressed in choroid plexus epithelial cells, play an important role in cerebrospinal fluid production and may be involved in the pathophysiology of age-dependent hydrocephalus. The finding that brain aquaporins expression is regulated by low oxygen tension led us to investigate how hypoxia and elevated levels of cerebral aquaporins may result in an increase in cerebrospinal fluid production that could be associated with a hydrocephalic condition. Here we have explored, in young and aged mice exposed to hypoxia, whether aquaporin-4 and aquaporin-1 participate in the development of age-related hydrocephalus. Choroid plexus, striatum, cortex and ependymal tissue were analyzed separately both for mRNA and protein levels of aquaporins. Furthermore, parameters such as total ventricular volume, intraventricular pressure, cerebrospinal fluid outflow rate, ventricular compliance and cognitive function were studied in wild type, aquaporin-1 and aquaporin-4 knock-out animals subjected to hypoxia or normoxia. Our data demonstrate that hypoxia is involved in the development of age-related hydrocephalus by a process that depends on aquaporin-4 channels as a main route for cerebrospinal fluid movement. Significant increases in aquaporin-4 expression that occur over the course of animal aging, together with a reduced cerebrospinal fluid outflow rate and ventricular compliance, contribute to produce more severe hydrocephalus related to hypoxic events in aged mice, with a notable impairment in cognitive function. These results indicate that physiological events and/or pathological conditions presenting with cerebral hypoxia/ischemia contribute to the development of chronic adult hydrocephalus.     

Dynamics of hydrocephalus: a physical approach

As brain ventricles lose their ability to regulate the cerebrospinal fluid (CSF) pressure, serious brain conditions collectively named hydrocephalus can appear. By modelling ventricular dynamics with the laws of physics, dynamical instabilities are evidenced, caused by either CSF transport dysregulations or abnormal properties of the elasticity of the ependyma. We show that these instabilities would lead, in most cases, to dilation of the ventricles, establishing a close connection to hydrocephalus, or in some other cases to a ventricular contraction as observed in the slit ventricle syndrome. Signs seem to indicate the possibility of phase transitions occurring as a result of these instabilities, which might have important clinical consequences, such as the inability to recover a healthy state. Even so, our dynamical approach could allow the development of a unified view of these complex intracranial conditions along with a classification that might be clinically relevant.     

Use of a novel double-sandwich enzyme-linked immunosorbent assay method for assaying chondroitin sulfate proteoglycans that bear 3-nitrotyrosine core protein modifications, a previously unrecognized proteoglycan modification in hydrocephalus

3-Nitrotyrosine is a useful marker for nitric oxide-mediated tissue injury. However, which proteins are preferred peroxynitrite modification targets is unclear. Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid of human neonates with hydrocephalus and may be a target for peroxynitrite modification. We examined (1). whether CSPG core protein can be modified by peroxynitrite in vitro; (2). to what degree in comparison to bovine serum albumin (BSA), the most commonly used nitrated protein standard; (3). whether nitrated CSPGs can be measured directly in biological samples; and (4). whether nitrated proteoglycan concentrations in cerebrospinal fluid correlate with disease. In vitro nitration of bovine aggrecan was performed by exposure to different peroxynitrite concentrations, and 3-nitrotyrosine products were measured. Bovine serum albumin (BSA) nitration was also performed in comparison. A larger percentage of tyrosine residues were nitrated in aggrecan than in BSA under all conditions tested. An enzyme-linked immunosorbent assay (ELISA) for 3-nitrotyrosine consistently overestimated aggrecan nitration when nitrated BSA was used as the standard. This is important as most current assays of nitration in biological samples use nitrated BSA as the standard. Therefore, if nitrated CPSGs were a substantial portion of the nitrated proteins in a sample, total nitrated protein content would be overestimated. Aggrecan retained its function of binding hyaluronic acid despite substantial nitration. A double-sandwich ELISA was developed for nitrated CSPGs in biological samples, using nitrated aggrecan as standard. [Nitrated CSPG] was found to be significantly elevated in preterm hydrocephalus cerebrospinal fluid (P<0.02), but correlated poorly with cerebrospinal fluid [nitric oxide] (P>0.069), suggesting that nitrated CSPG and NO levels may be independant markers of tissue injury. Peroxynitrite-mediated protein tyrosine nitration is a previously unrecognized modification of CSPGs, and may reflect level of brain injury in hydrocephalus.     

Ionic Composition of Cisternal CSF in Acute Respiratory Acidosis: Lack of Effect of Large Dose Bumetanide

Abstract: Sodium/chloride cotransport carrier is known to be involved in transepithelial fluid absorption and secretion in various tissues. Recent studies indicate that Na,K,2CI cotransport carrier also exists in the choroid plexus cells and inhibition of the carrier alters ionic composition of the choroidal tissue. In this study, we report the effects of large dose intravenous bumetanide, a potent inhibitor of Na,K,2CI carrier, on cisternal CSF ionic composition in acute respiratory acidosis in pentobarbital-anesthetized mechanically ventilated dogs. Renal pedicles were ligated to prevent bumetanide-induced diuresis. The experirnental group (Group II, n = 7) received 50 mg/kg of bumetanide intravenously and Group I (the control group, n = 7) received the vehicle. Analysis of serum and choroidal plexus tissue revealed bumetanide concentration of ∼10⁻-⁵ mol/L in Group II. During 5 h of acute respiratory acidosis in both groups, the mean Paco₂ increased ∼25 mm Hg, with comparable changes in CSF Pco₂. In both groups, CSF [HCO₃⁻] and [H⁺] increased ∼3 mEq/L and 20 nEq/L, respectively. Furthermore, changes in CSF [Na⁺], [K⁺], [Ca²⁺], [Mg²⁺], [CI⁻], and [Na⁺-CI⁻] were also similar and were not significantly different from each other. These data show that bumetanide, at the dose that inhibits NaCl cotransport carrier, does not significantly affect ionic composition of cisternal CSF.     

Breeding systems in Angiosperms: novel inferences from a new analytical approach

A novel analytical approach to classify breeding systems in Angiosperms combining statistical and conceptual criteria is proposed, which will allow to describe and compare available mating system data in an unified form. Four breeding system indexes (BSI) were combined: Index of Agamospermy, Index of Spontaneous Self-Pollination (ISSP), Index of Self-Fertility (ISF), and Index of Self-incompatibility. Their values, ranging from 0 to ∞, were analyzed using t tests to discriminate experimental breeding index values from 0 and 1.0. For each index, five discrete categories were described. The composite of the four BSI assigned to a species represents an integrated characterization of its reproductive system. A magnitude measure (M) describes the strength of each BSI assigned to a species. Indexes of inbreeding and outbreeding depression were also examined for each species. Published data from 1908 taxa were used to determine composite breeding systems. Frequencies of breeding index categories across the four BSI for the entire data set were determined. Non-agamospermous species dominated. Non-spontaneous self-pollinating and xenogamous species were the most frequent categories observed for ISSP and ISF indexes, respectively. The largest group of species examined was partially self-incompatible. Actual number of composite breeding systems inferred based on our analysis represents a small fraction (8.9 %) of all mathematically possible ones. Similarly, the observed combinations between results obtained from the four pollination tests and estimates of natural reproductive efficiency were only 22.1 % of all mathematically possible combinations. Within Angiosperms, there is a marked trend toward evolution of partial self-incompatibility with significant inbreeding depression.     

Gas Chromatographic-Mass Spectrometric Determination of myo-Inositol in Human Cerebrospinal Fluid

The concentration of free myo-inositol in CSF was determined with a gas chromatographic-mass spectrometric method using deuterated myo-inositol as an internal standard after conversion to the hexa-O-acetyl derivative with acetic anhydride and pyridine. Twenty microliters of CSF is sufficient for the analysis which has a coefficient of variation of 9%. Identical analytical results were obtained on two different mass numbers. Schizophrenic patients were compared with healthy control persons. In addition, patients with rheumatoid arthritis or with neurological illnesses were studied. No consistent differences related to the illness could be found. The mean concentration of myo-inositol was about 25 micrograms/ml. Treatment of schizophrenic patients with chlorpromazine or sulpiride had no significant effect on the concentration of myo-inositol in CSF.     

Neural precursors express multiple chondroitin sulfate proteoglycans, including the lectican family

Chondroitin sulfate proteoglycans (CSPGs) abnormally accumulate in cerebrospinal fluid (CSF) of both human neonates with preterm hydrocephalus, and P8 hydrocephalic mice. We hypothesized CSF CSPGs are synthesized by neural precursors, separated from ventricular CSF by ependyma, which is often disrupted in hydrocephalus. Western blotting demonstrates that neural precursors cultured as neurospheres secrete CSPGs (> 30 microg/ml) into their media which appear to be very similar to these CSF CSPGs. Some CSPGs bear the stage-specific embryonic antigen-1 (ssea-1), associated with embryonic/neural stem cells. Neurospheres transcribe many CSPG genes, including the entire aggrecan/lectican family, phosphacan, and tenascin. Phosphacan can be detected in media by Western blotting. Aggrecan can be detected in media after purification using hyaluronic acid affinity chromatography. During differentiation, neurospheres downregulate CSPGs. This is the first report to show that proliferating neural precursors synthesize lecticans, including aggrecan, which are downregulated with differentiation. These observations suggest novel links between CSPGs and CNS precursor biology.     

An empirical approach to characterizing trunk muscle coactivation using simulation input modeling techniques

Accurately describing trunk muscle coactivation is fundamental to quantifying the spine reaction forces that occur during lifting tasks and has been the focus of a great deal of research in the spine biomechanics literature. One limitation of previous approaches has been a lack of consideration given to the variability in these coactivation strategies. The research presented in this paper is an empirical approach to quantifying and modeling trunk muscle coactivation using simulation input modeling techniques. Electromyographic (EMG) data were collected from 28 human subjects as they performed controlled trunk extension exertions. These exertions included isokinetic (10 and 45°/s) and constant acceleration (50°/s/s) trunk extensions in symmetric and asymmetric (30°) postures at two levels of trunk extension moment (30 and 80 Nm). The EMG data were collected from the right and left pairs of the erector spinae, latissimus dorsi, rectus abdominis, external obliques and internal obliques. Each subject performed nine repetitions of each combination of independent variables. The data collected during these trials were used to develop marginal distributions of trunk muscle activity as well as a 10×10 correlation matrix that described how the muscles cooperated to produce these extension torques. These elements were then combined to generate multivariate distributions describing the coactivation of the trunk musculature. An analysis of these distributions revealed that increases in extension moment, extension velocity and sagittal flexion angle created increases in both the mean and the variance of the distributions of the muscular response, while increases in the rate of trunk extension acceleration decreased both the mean and variance of the distributions of activity across all muscles considered. Increases in trunk asymmetry created a decrease in mean of the ipsi–lateral erector spinae and an increase in the mean of all other muscles considered, but there was little change in the variance of these distributions as a function of asymmetry.     

High-pressure freezing combined with in vivo-DAB-cytochemistry: A novel approach for studies of endocytic compartments

Methods for fine structural and functional analyses of complex and dynamic cell compartments must ensure high temporal resolution together with an excellent fine structural preservation. High-pressure freezing followed by freeze-substitution, and resin embedding is state of the art but its use is limited in combination with preembedding cytochemical techniques. Here we show a new approach for the exploration of compartments of the endocytosis system, which combines high-pressure freezing with peroxidase-catalyzed cytochemistry, thus using the potencies of both synergistically. Uptake of horseradish peroxidase-labeled molecules is followed by in vivo-staining and immobilization of endocytic compartments by generation of diaminobenzidine precipitates. Subsequently, the specimens are high pressure frozen, freeze-substituted, and embedded in resin. The excellent fine structural preservation, together with the high temporal resolution, and differentiating visualization of endocytic compartments qualify the new approach for morpho-functional studies of the complex and dynamic components of the endocytosis system involved in physiologic and pathologic cellular traffic, and in routes utilized in drug targeting strategies. The distinct appearances of membranes and reactive compartments provide optimal conditions for 3D-analyses by electron tomography allowing to discern subtle details of the complex 3D-structures of endocytic compartments.     

Structure-based design of a novel peptide inhibitor of HIV-1 integrase: a computer modeling approach

The insertion of viral DNA into the host chromosome is an essential step in the replication of HIV-1, and is carried out by an enzyme, HIV-1 integrase (IN). Since the latter has no human cellular counterpart, it is an attractive target for antiviral drug design. Several IN inhibitors having activities in the micromolar range have been reported to date. However, no clinically useful inhibitors have yet been developed. Recently reported diketo acids represent a novel and selective class of IN inhibitors. These are the only class which appear to selectively target integrase and two of the inhibitors, L-708,906 and L-731,988, are the most potent inhibitors of preintegration complexes described to date.The X-ray crystal structure of the IN catalytic domain complexed with a diketo acid derivative inhibitor, 5CITEP, has recently been determined. Although the structure is of great value as a platform for drug design, experimental data suggest that crystal packing effects influence the observed inhibitor position. This has been confirmed by computational docking studies using the latest version (3.0) of the AutoDock program, which has been shown to give results largely consistent with available experimental data. Using AutoDock 3.0 and SYBYL6.6 we have modeled the complexes of IN with the diketo acid inhibitors so as to identify the enzyme binding site. In the quest for novel, potent and selective small molecule inhibitors, we present here a new approach to peptide inhibitor design using a, b- unsaturated (dehydro) residues, which confer a unique conformation on a peptide sequence. Based on the above models, we selected a tetrapeptide sequence containing a dehydro-Phe residue, which was found to have an open conformation as ascertained from its X-ray crystal structure. Docking results on this peptide led us to propose a modification at the C-terminal end. The modified peptide was found to dock in a similar position as the diketo acid inhibitors and was predicted to have a comparable potency.     

A scenario-based approach to modeling development: a prototype model of C. elegans vulval fate specification

Studies of developmental biology are often facilitated by diagram “models” that summarize the current understanding of underlying mechanisms. The increasing complexity of our understanding of development necessitates computational models that can extend these representations to include their dynamic behavior. Here we present a prototype model of Caenorhabditis elegans vulval precursor cell fate specification that represents many processes crucial for this developmental event but that are hard to integrate using other modeling methodologies. We demonstrate the integrative capabilities of our methodology by comprehensively incorporating the contents of three seminal papers, showing that this methodology can lead to comprehensive models of developmental biology. The prototype computational model was built and is run using a language (Live Sequence Charts) and tool (the Play-Engine) that facilitate the same conceptual processes biologists use to construct and probe diagram-type models. We demonstrate that this modeling approach permits rigorous tests of mutual consistency between experimental data and mechanistic hypotheses and can identify specific conflicting results, providing a useful approach to probe developmental systems.     

A hierarchical whole-body modeling approach elucidates the link between in Vitro insulin signaling and in Vivo glucose homeostasis

Type 2 diabetes is a metabolic disease that profoundly affects energy homeostasis. The disease involves failure at several levels and subsystems and is characterized by insulin resistance in target cells and tissues (i.e. by impaired intracellular insulin signaling). We have previously used an iterative experimental-theoretical approach to unravel the early insulin signaling events in primary human adipocytes. That study, like most insulin signaling studies, is based on in vitro experimental examination of cells, and the in vivo relevance of such studies for human beings has not been systematically examined. Herein, we develop a hierarchical model of the adipose tissue, which links intracellular insulin control of glucose transport in human primary adipocytes with whole-body glucose homeostasis. An iterative approach between experiments and minimal modeling allowed us to conclude that it is not possible to scale up the experimentally determined glucose uptake by the isolated adipocytes to match the glucose uptake profile of the adipose tissue in vivo. However, a model that additionally includes insulin effects on blood flow in the adipose tissue and GLUT4 translocation due to cell handling can explain all data, but neither of these additions is sufficient independently. We also extend the minimal model to include hierarchical dynamic links to more detailed models (both to our own models and to those by others), which act as submodules that can be turned on or off. The resulting multilevel hierarchical model can merge detailed results on different subsystems into a coherent understanding of whole-body glucose homeostasis. This hierarchical modeling can potentially create bridges between other experimental model systems and the in vivo human situation and offers a framework for systematic evaluation of the physiological relevance of in vitro obtained molecular/cellular experimental data.     

Clusterin in cerebrospinal fluid: analysis of carbohydrates and quantification of native and glycosylated forms

Clusterin is suggested to be involved in the pathogenesis of Alzheimer's disease. Clusterin expression is increased in brain tissue in affected regions of Alzheimer patients, and intense clusterin staining is found in both senile plaques and in neuronal and glia cells. In contrast, the cerebrospinal fluid level of clusterin in Alzheimer patients has, thus far, been found unchanged. Clusterin is a glycosylated protein, and an alteration of its glycosylation in Alzheimer's disease might influence accurate quantification in cerebrospinal fluid through interference of antibody binding to the protein. Using enzymatic deglycosylation of clusterin isolated from cerebrospinal fluid, we found that the carbohydrates attached to clusterin were of the N-linked type and sialic acids. Based on this finding, cerebrospinal fluid samples from Alzheimer patients (n = 99) and controls (n = 39) were analysed. The samples were treated with peptide: N-glycanase F, cleaving off N-linked carbohydrates, and clusterin was quantified before and after deglycosylation using a new sandwich enzyme-linked immunosorbent assay. Clusterin was significantly increased in Alzheimer patients, in both native (7.17 ± 2.43 AU versus 5.73 ± 2.09 AU; p = 0.002), and deglycosylated samples (12.19 ± 5.00 AU versus 9.68 ± 4.38 AU; p = 0.004). Deglycosylation led to increased measured levels of clusterin by 70% (p < 0.001) in Alzheimer patients and 67% (p < 0.001) in controls. These findings indicate that glycosylation of proteins may interfere with their quantification. The results show that clusterin is significantly increased in cerebrospinal fluid from Alzheimer patients as a group, supporting that clusterin might be involved in the pathogenesis of Alzheimer's disease. However, the individual clusterin levels overlap between the two groups, and thus cerebrospinal fluid clusterin measurement is not suitable as a biochemical marker in the diagnosis of Alzheimer's disease.     

Successional state dynamics: A novel approach to modeling nonequilibrium foodweb dynamics

Communities and ecosystems are often far from equilibrium, but our understanding of nonequilibrium dynamics has been hampered by a paucity of analytical tools. Here I describe a novel approach to modeling seasonally forced food webs, called “successional state dynamics” (SSD). It is applicable to communities where species dynamics are fast relative to the external forcing, such as plankton and other microbes, diseases, and some insect communities. The approach treats succession as a series of state transitions driven by both the internal dynamics of species interactions and external forcing. First, I motivate the approach with numerical solutions of a seasonally forced predator-prey model. Second, I describe how to set up and analyze an SSD model. Finally, I apply the techniques to three additional models of two-species interactions: resource competition (r-K selection), facilitation, and flip-flop competition (where the competitive hierarchy alternates over time). This approach allows easy and thorough exploration of how dynamics depend on the environmental forcing regime, and uncovers unexpected phenomena such as multiple stable annual trajectories and year-to-year irregularity in successional trajectories (chaos).     

一种Beagle犬腰椎穿刺置管术介绍

目的建立一种新的Beagle犬脑脊液连续留取的方法。方法通过氯胺酮麻醉进行腰椎穿刺后置管可连续留取脑脊液。结果腰椎穿刺置管后成功连续留取清亮脑脊液超过48h。结论腰椎穿刺置管连续留取脑脊液简单、有效。     

Effects of fluid structure interaction in a three dimensional model of the spinal subarachnoid space

It is unknown whether spinal cord motion has a significant effect on cerebrospinal fluid (CSF) pressure and therefore the importance of including fluid structure interaction (FSI) in computational fluid dynamics models (CFD) of the spinal subarachnoid space (SAS) is unclear. This study aims to determine the effects of FSI on CSF pressure and spinal cord motion in a normal and in a stenosis model of the SAS. A three-dimensional patient specific model of the SAS and spinal cord were constructed from MR anatomical images and CSF flow rate measurements obtained from a healthy human being. The area of SAS at spinal level T4 was constricted by 20% to represent the stenosis model. FSI simulations in both models were performed by running ANSYS CFX and ANSYS Mechanical in tandem. Results from this study show that the effect of FSI on CSF pressure is only about 1% in both the normal and stenosis models and therefore show that FSI has a negligible effect on CSF pressure.