Brain Interstitial Fluid Research Articles

Nanoparticulated Quercetin in Combating Age Related Cerebral Oxidative Injury

Reactive oxygen species e.g. O(2)(*-), H(2)O(2) and *OH generated by the induction of oxidative stress exert a potential threat on the activity of endogenous antioxidant enzymes and substantially influence the aging process and age-dependant neuropathology. Chemical antioxidant is almost ineffective in protecting neuronal cells from oxidative damage as Blood Brain Barrier exists in between blood and brain interstitial fluid that restricts undegradable influx from the circulation into cerebral region. Quercetin (QC), a flavonoidal antioxidant is known as a potent antioxidant for its polyphenolic configuration. Formulation of QC in polylactide nanocapsule has been done and the efficacy of this vesicular flavonoid has been tested against cerebral ischemia induced oxidative damage in young and old rat brains. Antioxidant potential of QC loaded in nanocapsule (QC 7.2 mmol/kg b.wt., size 50 nm) was investigated by an in vivo model of cerebral ischemia and reperfusion on Sprague Dawley young (2 months, b.wt. 160-180 g) and aged (20 months, b.wt. 415-440 g) rats. Diene level, the index of lipid peroxidation and GSSG/GSH ratio were found to be higher in normal aged, compared to normal young rat brain. Endogenous antioxidants activities were lower in aged rat brain compared to young. Further reduction of these antioxidants were observed in aged rat brain by the induction of cerebral ischemia - reperfusion. Nanocapsule encapsulated QC treatment resulted a significant protection to endogenous antioxidant enzymes against ischemia induced oxidative damage in neuronal cells of young and old rats.

Amyloid-β Dynamics Correlate with Neurological Status in the Injured Human Brain

The amyloid-beta peptide (Abeta) plays a central pathophysiological role in Alzheimer's disease, but little is known about the concentration and dynamics of this secreted peptide in the extracellular space of the human brain. We used intracerebral microdialysis to obtain serial brain interstitial fluid (ISF) samples in 18 patients who were undergoing invasive intracranial monitoring after acute brain injury. We found a strong positive correlation between changes in brain ISF Abeta concentrations and neurological status, with Abeta concentrations increasing as neurological status improved and falling when neurological status declined. Brain ISF Abeta concentrations were also lower when other cerebral physiological and metabolic abnormalities reflected depressed neuronal function. Such dynamics fit well with the hypothesis that neuronal activity regulates extracellular Abeta concentration.

Cerebrospinal fluid biomarkers of neurodegeneration in chronic neurological diseases

Chronic neurological diseases (CND) like amyotrophic lateral sclerosis (ALS), dementia or multiple sclerosis (MS) share a chronic progressive course of disease that frequently leads to the common pathological pathway of neurodegeneration, including neuroaxonal damage, apoptosis and gliosis. There is an ongoing search for biomarkers that could support early diagnosis of CND and help to identify responders to interventions in therapeutic treatment trials. Cerebrospinal fluid (CSF) is a promising source of biomarkers in CND, since the CSF compartment is in close anatomical contact with the brain interstitial fluid, where biochemical changes related to CND are reflected. We review recent advances in CSF biomarkers research in CND and thereby focus on markers associated with neurodegeneration.

S2-04-04: Rational therapeutic strategies for modifying Alzheimer's disease: Abeta oligomers as the validated target

In 1984 we described the oligomerization of Aβ and some of the biochemical factors which affect its stability. Subsequently, we found that metal ions such as Zn2+ and Cu2+ play a major role in the oligomerization process, and that the soluble oligomeric species of Aβ are the principal determinants of the degree of neurodegeneration. We next identified compounds which were capable of interfering with the metal-induced oligomerization of Aβ. Prana Biotechnology (PBT) was founded to commercialize these discoveries, and the first compound (clioquinol – CQ) was shown to have favorable preclinical properties and some efficacy in an early Phase 2 clinical trial. Further medicinal chemistry on the 8-OH quinoline scaffold yielded a much-improved compound, PBT2, with high brain penetrability. CQ and PBT2 are efficient at inhibiting Zn2+ and Cu2+-induced Aβ oligomerization and Cu2+-induced dityrosine formation. In transgenic mouse AD models, CQ and PBT2 effectively: lower soluble oligomer and insoluble Aβ levels in brain interstitial fluid dialysate and Aβ plaque levels; reverse Aβ-induced inhibition of LTP in hippocampal slices; improve the behavioral phenotype in the Morris Water Maze; lower tau, and increase synaptophysin levels. PBT2 has now been taken through early clinical development. In a recently completed Phase 2 study, the compound was found to be safe at 50 and 250mg per day for 12 weeks. CSF Aβ42 was significantly reduced at the higher dose, with a significant dose-effect. Two tests of cognitive executive function also showed significant improvements at the higher dose. Aβ-amyloid neuroimaging using PET offer the tools for monitoring the efficacy of Aβ-directed drug interventions, and shows a highly significant correlation of Aβ-amyloid burden with cognitive impairment during the evolution of the preclinical phases of AD. Collectively, these data provide evidence that orally bioavailable drugs designed to inhibit toxic oligomeric species of Aβ in human subjects with Alzheimer's disease have the potential to modify the course of the illness.

P4-081: Diurnal fluctuation of amyloid-β in the brain interstitial fluid of young Tg2576 mice

P4-081: Diurnal fluctuation of amyloid-β in the brain interstitial fluid of young Tg2576 mice

Endocytosis Is Required for Synaptic Activity-Dependent Release of Amyloid-β In Vivo

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Entry of Oximes into the Brain: A Review

The passage of hydrophilic drugs, such as oxime acetylcholinesterase reactivators, into the central nervous system is restricted by the blood-brain and the blood-cerebrospinal fluid barriers. The present review summarizes morphological and functional properties of the blood-brain barrier, blood-cerebrospinal fluid barrier and cerebrospinal fluid-brain interface and reviews the existing data on brain entry of oximes. Due to the virtual absence of transcytosis, lack of fenestrations and unique properties of tight junctions in brain endothelial cells, the blood-brain barrier only allows free diffusion of small lipophilic molecules. Various carriers transport hydrophilic compounds and extrude potentially toxic xenobiotics. The blood-cerebrospinal fluid barrier is formed by the choroid plexus epithelium, whose tight junctions are more permeable than those of brain endothelial cells. The major function of plexus epithelium cells is active transport of ions for the production of the cerebrospinal fluid. The cerebrospinal fluid-brain interface is not a biological barrier and allows free diffusion. However, in contrast to passage via the blood-brain barrier or the blood-cerebrospinal fluid barrier, direct penetration from the cerebrospinal fluid into the brain is very slow, since much longer distances have to be covered. A bulk flow of brain interstitial fluid and cerebrospinal fluid speeds up exchange between these two fluid compartments. Oximes, by reactivating acetylcholinesterase, are important adjunct therapeutics in organophosphate poisoning. They are very hydrophilic and therefore cannot diffuse freely into the central nervous system. Changes in brain acetylcholinesterase activity, oxime concentration and some biological effects elicited by oxime administration in the periphery indicate, however, that oximes can gain access to the brain to a certain degree, probably by carrier-mediated transport, reaching in the brain about 4-10% of their respective plasma levels. The clinical relevance of this effect is hotly debated. Possible strategies to improve brain penetration of oximes are discussed.

Kv1 and Kir2 potassium channels are expressed in rat brain endothelial cells

The endothelial cells of the brain microvasculature, which constitute the blood-brain barrier, secrete K+ into brain interstitial fluid. K+ channels are predicted to have a central role to play in this process. The aim of the following study was to characterise K+ channels in primary cultures of endothelial cells isolated from rat brain microvessels by whole-cell patch clamp and real-time polymerase chain reaction. In the 4 h after plating, the rat brain endothelial cells expressed predominantly a depolarisation-activated delayed-rectifying outward K+ conductance and a time-independent inwardly rectifying K+ conductance prominent at hyperpolarising potentials. The outward current was inhibited by 1 mM 4-aminopyridine (4AP), 10 nM margatoxin and 100 nM dendrotoxin-K, indicating the involvement of Kv1 channels. The half maximal activation voltage and time constants of activation and inactivation of the 4AP-sensitive current were similar to Kv1.3. The inwardly rectifying conductance was inhibited by Ba2+ in a dose- and voltage-dependent fashion; the kinetics of which resembled Kir2 channels. Quantification of messenger ribonucleic acid transcripts revealed Kv1.3>1.2=1.4=1.5=1.6 and Kir2.1=2>2.3. In current-clamp experiments, both 4AP and Ba2+ depolarised the membrane potential. In conclusion, rat brain endothelial cells express Kv1 and Kir2 K+ channels, both of which participate in setting membrane potential and could mediate K+ secretion into the brain interstitial fluid.

Loss of Neprilysin Function Promotes Amyloid Plaque Formation and Causes Cerebral Amyloid Angiopathy

Cerebral deposition of the amyloid beta protein (Abeta), an invariant feature of Alzheimer's disease, reflects an imbalance between the rates of Abeta production and clearance. The causes of Abeta elevation in the common late-onset form of Alzheimer's disease (LOAD) are largely unknown. There is evidence that the Abeta-degrading protease neprilysin (NEP) is down-regulated in normal aging and LOAD. We asked whether a decrease in endogenous NEP levels can prolong the half-life of Abeta in vivo and promote development of the classic amyloid neuropathology of Alzheimer's disease. We examined the brains and plasma of young and old mice expressing relatively low levels of human amyloid precursor protein and having one or both NEP genes silenced. NEP loss of function 1) elevated whole-brain and plasma levels of human Abeta(40) and Abeta(42), 2) prolonged the half-life of soluble Abeta in brain interstitial fluid of awake animals, 3) raised the concentration of Abeta dimers, 4) markedly increased hippocampal amyloid plaque burden, and 5) led to the development of amyloid angiopathy. A approximately 50% reduction in NEP levels, similar to that reported in some LOAD brains, was sufficient to increase amyloid neuropathology. These findings demonstrate an important role for proteolysis in determining the levels of Abeta and Abeta-associated neuropathology in vivo and support the hypothesis that primary defects in Abeta clearance can cause or contribute to LOAD pathogenesis.

In Vitro Methods for Estimating Unbound Drug Concentrations in the Brain Interstitial and Intracellular Fluids

Concentrations of unbound drug in the interstitial fluid of the brain are not rapidly measured in vivo. Therefore, measurement of total drug levels, i.e., the amount of drug per gram of brain, has been a common but unhelpful practice in drug discovery programs relating to central drug effects. This study was designed to evaluate in vitro techniques for faster estimation of unbound drug concentrations. The parameter that relates the total drug level and the unbound interstitial fluid concentration is the unbound volume of distribution in the brain (V(u,brain)). It was measured in vitro for 15 drugs using brain slice uptake and brain homogenate binding methods. The results were validated in vivo by comparison with V(u,brain) microdialysis results. The slice method results were within a 3-fold range of the in vivo results for all but one compound, suggesting that this method could be used in combination with total drug levels to estimate unbound interstitial fluid concentrations within reasonable limits. Although successful in 10 of 15 cases, the brain homogenate binding method failed to estimate the V(u,brain) of drugs that reside predominantly in the interstitial space or compounds that are accumulated intracellularly. Use of the simple methods described in this article will 1) allow quantification of active transport at the blood-brain barrier in vivo, 2) facilitate the establishment of a relationship between in vitro potency and in vivo activity for compounds acting on central nervous system targets, and 3) provide information on intracellular concentrations of unbound drug.

Acute stress increases interstitial fluid amyloid-β via corticotropin-releasing factor and neuronal activity

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is critical in the pathogenesis of Alzheimer's disease. Abeta is produced by neurons and released into the brain interstitial fluid (ISF), a process regulated by synaptic activity. To determine whether behavioral stressors can regulate ISF Abeta levels, we assessed the effects of chronic and acute stress paradigms in amyloid precursor protein transgenic mice. Isolation stress over 3 months increased Abeta levels by 84%. Similarly, acute restraint stress increased Abeta levels over hours. Exogenous corticotropin-releasing factor (CRF) but not corticosterone mimicked the effects of acute restraint stress. Inhibition of endogenous CRF receptors or neuronal activity blocked the effects of acute stress on Abeta. Thus, behavioral stressors can rapidly increase ISF Abeta through neuronal activity in a CRF-dependent manner, and the results suggest a mechanism by which behavioral stress may affect Alzheimer's disease pathogenesis.

The Origin of Deoxynucleosides in Brain: Implications for the Study of Neurogenesis and Stem Cell Therapy

Detection of DNA synthesis in brain employing ((3)H)thymidine (((3)H)dT) or bromo deoxyuridine (BrdU) is widely used as a measure of the "birth" of cells in brain development, adult neurogenesis and neuronal stem cell replacement strategies. However, recent studies have raised serious questions about whether this methodology adequately measures the "birth" of cells in brain either quantitatively or in an interpretable way in comparative studies, or in stem cell investigations. To place these questions in perspective, we review deoxynucleoside synthesis and pharmacokinetics focusing on the barriers interfacing the blood-brain (cerebral capillaries) and blood-cerebrospinal fluid (choroid plexus), and the mechanisms, molecular biology and location of the deoxynucleoside transport systems in the central nervous system. Brain interstitial fluid and CSF nucleoside homeostasis depend upon the activity of concentrative nucleoside transporters (CNT) on the 'central side' of the barrier cells and equilibrative nucleoside transporters (ENT) on their 'plasma side.' With this information about nucleoside transporters, blood/CSF concentrations and metabolic pathways, we discuss the assumptions and weaknesses of using ((3)H)dT or BrdU methodologies alone for studying DNA synthesis in brain in the context of neurogenesis and potential stem cell therapy. We conclude that the use of ((3)H)dT and/or BrdU methodologies can be useful if their limitations are recognized and they are used in conjunction with independent methods.

Retro-convection enhanced delivery to increase blood to brain transfer of macromolecules

A retro-convection enhanced delivery (R-CED) method has been developed to improve the entry of intravenously administered therapeutics within solid brain tumors. R-CED uses an osmotic gradient to withdraw brain interstitial fluid (ISF) in a controlled manner via an implanted microdialysis catheter. Withdrawal of ISF increases the local tissue specific gravity in normal brain and increases twofold the extravasation of intravenous Evans blue (EB) albumin in normal brain and in an orthotopic 9L tumor. R-CED also increases the extravasation of 70 nm fluorescent liposomes fivefold in the 9L tumor. Thus the transmembrane osmotic gradient induces movement of substances in the blood into the tissue parenchyma. Following probe removal, the magnitude of the R-CED effect on EB-albumin extravasation decreases to control values within 1.5 h in normal brain; however, the effect persists beyond 6 h in the tumor. There was no evidence of histologic damage to the neurons at either 6 h or 2 weeks after R-CED. These studies establish the feasibility of applying R-CED to increase the distribution of systemically administered drugs in both the normal tissue–tumor margin as well as in the central tumor core, holding forth the possibility of improved antitumor drug efficacy.

Matrix Metalloproteinases Expressed by Astrocytes Mediate Extracellular Amyloid-β Peptide Catabolism

It has been postulated that the development of amyloid plaques in Alzheimer's disease (AD) may result from an imbalance between the generation and clearance of the amyloid-beta peptide (Abeta). Although familial AD appears to be caused by Abeta overproduction, sporadic AD (the most prevalent form) may result from impairment in clearance. Recent evidence suggests that several proteases may contribute to the degradation of Abeta. Furthermore, astrocytes have recently been implicated as a potential cellular mediator of Abeta degradation. In this study, we examined the possibility that matrix metalloproteinases (MMPs), proteases known to be expressed and secreted by astrocytes, could play a role in extracellular Abeta degradation. We found that astrocytes surrounding amyloid plaques showed enhanced expression of MMP-2 and MMP-9 in aged amyloid precursor protein (APP)/presenilin 1 mice. Moreover, astrocyte-conditioned medium (ACM) degraded Abeta, lowering levels and producing several fragments after incubation with synthetic human Abeta(1-40) and Abeta(1-42). This activity was attenuated with specific inhibitors of MMP-2 and -9, as well as in ACM derived from mmp-2 or -9 knock-out (KO) mice. In vivo, significant increases in the steady-state levels of Abeta were found in the brains of mmp-2 and -9 KO mice compared with wild-type controls. Furthermore, pharmacological inhibition of the MMPs with N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide (GM 6001) increased brain interstitial fluid Abeta levels and elimination of half-life in APPsw mice. These results suggest that MMP-2 and -9 may contribute to extracellular brain Abeta clearance by promoting Abeta catabolism.

P4-004: Microdialysis reveals β-amyloid levels in brain interstitial fluid regulated by γ-secretase and neprilysin inhibitors as well as overexpression of β-secretase in vivo

P4-004: Microdialysis reveals β-amyloid levels in brain interstitial fluid regulated by γ-secretase and neprilysin inhibitors as well as overexpression of β-secretase in vivo

Effects of chronic ethanol administration on brain interstitial fluid levels of Methionine-enkephalin as measured by microdialysis in vivo

The level of Met-enkephalin in the brain is inversely correlated with ethanol consumption and is controlled partially through efflux activity of peptide transport system-1 (PTS-1) at the blood–brain barrier (BBB). Prolonged alcohol drinking can perturb aspects of this system, including a loss of control of Met-enkephalin levels at the transcriptional and translational levels, and impaired release of Met-enkephalin from tissue sources. Met-enkephalin levels in whole brain homogenates often first paradoxically increase after a few days of ethanol drinking and then decrease with the development of physical dependence. Which of those various changes drives the others is unclear. To clarify these interactions, we here determined the levels of Met-enkephalin in striatal interstitial fluid (ISF) by microdialysis, striatal tissue homogenates, and serum after chronic ethanol treatment and alcohol withdrawal. Mice received ethanol (5%) in liquid diet for 7 days (ethanol-treated) and others withdrawn for a day following 7-day treatment (withdrawal). There was a significant ( P < 0.05) difference in the levels of Met-enkephalin in striatal microdialysate between the control (79.1 ± 5.9 pg/ml) and ethanol-treated group (94.9 ± 4.3 pg/ml), which was lost by withdrawing ethanol (83.9 ± 3.8 pg/ml). In contrast, ethanol treatment did not affect Met-enkephalin levels in the striatal tissue. In the ethanol-treated group, there was a significant ( P < 0.05) reduction of the levels of Met-enkephalin in serum to 70.5% of control levels. This decrease was restored to the level of control by withdrawing ethanol. These reversible changes in ISF and serum are readily explained by the known changes in the efflux activity of PTS-1 at the BBB.

Synaptic Activity Regulates Interstitial Fluid Amyloid-β Levels In Vivo

Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is central to Alzheimer's disease pathogenesis. Abeta aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Abeta levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Abeta levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Abeta metabolism and ultimately region-specific Abeta deposition. The findings also have important implications for treatment development.

Aging rat brain: a model for analyzing interactions among CSF dynamics, ventriculomegaly and the β-amyloid retention of alzheimer's disease

Background Age-associated disruptions in brain barrier systems (including choroid plexus) lead to multiple problems for CSF turnover and brain interstitial fluid composition. Age is a great risk factor for Alzheimer's disease (AD). We have presented evidence that beta-amyloid (Aβ) retention in AD is linked to decreased CSF turnover and reduced Aβ transport out of human brain. It is also known that CSF formation is reduced in aged rats (Preston et al.).

P-glycoprotein deficiency at the blood-brain barrier increases amyloid-beta deposition in an Alzheimer disease mouse model.

Accumulation of amyloid-beta (Abeta) within extracellular spaces of the brain is a hallmark of Alzheimer disease (AD). In sporadic, late-onset AD, there is little evidence for increased Abeta production, suggesting that decreased elimination from the brain may contribute to elevated levels of Abeta and plaque formation. Efflux transport of Abeta across the blood-brain barrier (BBB) contributes to Abeta removal from the brain. P-glycoprotein (Pgp) is highly expressed on the luminal surface of brain capillary endothelial cells and contributes to the BBB. In Pgp-null mice, we show that [I]Abeta40 and [I]Abeta42 microinjected into the CNS clear at half the rate that they do in WT mice. When amyloid precursor protein-transgenic (APP-transgenic) mice were administered a Pgp inhibitor, Abeta levels within the brain interstitial fluid significantly increased within hours of treatment. Furthermore, APP-transgenic, Pgp-null mice had increased levels of brain Abeta and enhanced Abeta deposition compared with APP-transgenic, Pgp WT mice. These data establish a direct link between Pgp and Abeta metabolism in vivo and suggest that Pgp activity at the BBB could affect risk for developing AD as well as provide a novel diagnostic and therapeutic target.

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.