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Brain pericytes from stress-susceptible pigs increase blood-brain barrier permeability in vitro
Background:
The function of pericytes remains questionable but with improved cultured technique and theuse of genetically modified animals, it has become increasingly clear that pericytes are anintegral part of blood-brain barrier (BBB) function, and the involvement of pericytedysfunction in certain cerebrovascular diseases is now emerging. The porcine stresssyndrome (PSS) is the only confirmed, homologous model of malignant hyperthermia (MH)in veterinary medicine. Affected animals can experience upon slaughter a range of symptoms,including skeletal muscle rigidity, metabolic acidosis, tachycardia and fever, similar to thehuman syndrome. Symptoms are due to an enhanced calcium release from intracellularstores. These conditions are associated with a point mutation in ryr1/hal gene, encoding theryanodine receptor, a calcium channel. Important blood vessel wall muscle modificationshave been described in PSS, but potential brain vessel changes have never been documentedin this syndrome.
Methods:
In the present work, histological and ultrastructural analyses of brain capillaries from wildtype and ryr1 mutated pigs were conducted to investigate the potential impairment ofpericytes, in this pathology. In addition, brain pericytes were isolated from the three porcinegenotypes (wild-type NN pigs; Nn and nn pigs, bearing one or two (n) mutant ryr1/halalleles, respectively), and tested in vitro for their influence on the permeability of BBBendothelial monolayers.
Results:
Enlarged perivascular spaces were observed in ryr1-mutant samples, corresponding to apartial or total detachment of the astrocytic endfeet. These spaces were electron lucent andsometimes filled with lipid deposits and swollen astrocytic feet. At the ultrastructural level,brain pericytes did not seem to be affected because they showed regular morphology andcharacteristics, so we aimed to check their ability to maintain BBB properties in vitro. Ourresults indicated that pericytes from the three genotypes of pigs had differing influences onthe BBB. Unlike pericytes from NN pigs, pericytes from Nn and nn pigs were not able tomaintain low BBB permeability.
Conclusions:
Electron microscopy observations demonstrated brain capillary modifications in PSScondition, but no change in pericyte morphology. Results from in vitro experiments suggestthat brain pericytes from ryr1 mutated pigs, even if they are not affected by this condition atthe ultrastructural level, are not able to maintain BBB integrity in comparison with pericytesfrom wild-type animals.
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Blood-brain barrier disruption in CCL2 transgenic mice during pertussis toxin-induced brain inflammation
Background:
The chemokine CCL2 has an important role in the recruitment of inflammatory cells into the central nervous system (CNS). A transgenic mouse model that overexpresses CCL2 in the CNS shows an accumulation of leukocytes within the perivascular space surrounding vessels, which infiltrate into the brain parenchyma following the administration of pertussis toxin (PTx).
Methods:
This study uses contrast-enhanced magnetic resonance imaging (MRI) to quantify the extent of blood-brain barrier (BBB) disruption in this model pre- and post-PTx administration compared to wild type mice. Contrast-enhanced MR images were obtained before and 1, 3, and 5 days after PTx injection in each animal. After the final imaging session fluorescent dextran tracers were administered intravenously to each mouse and brains were examined histologically for cellular infiltrates, BBB leakage and tight junction protein.
Results:
BBB breakdown, defined as a disruption of both the endothelium and glia limitans, was found only in CCL2 transgenic mice following PTx administration seen on MR images as focal areas of contrast enhancement and histologically as dextrans leaking from blood vessels. No evidence of disruption in endothelial tight junctions was observed.
Conclusion:
Genetic and environmental stimuli were needed to disrupt the integrity of the BBB in this model of neuroinflammation.
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Visualisation of cerebrospinal fluid flow patterns in albino Xenopus larvae in vivo
Background:
It has long been known that cerebrospinal fluid (CSF), its composition and flow, play an important part in normal brain development, and ependymal cell ciliary beating as a possible driver of CSF flow has previously been studied in mammalian fetuses in vitro. Lower vertebrate animals are potential models for analysis of CSF flow during development because they are oviparous. Albino Xenopus laevis larvae are nearly transparent and have a straight, translucent brain that facilitates the observation of fluid flow within the ventricles. The aim of these experiments was to study CSF flow and circulation in vivo in the developing brain of living embryos, larvae and tadpoles of Xenopus laevis using a microinjection technique.
Methods:
The development of Xenopus larval brain ventricles and the patterns of CSF flow were visualised after injection of quantum dot nanocrystals and polystyrene beads (3.1 or 5.8 μm in diameter) into the fourth cerebral ventricle at embryonic/larval stages 30-53.
Results:
The fluorescent nanocrystals showed the normal development of the cerebral ventricles from embryonic/larval stages 38 to 53. The polystyrene beads injected into stage 47-49 larvae revealed three CSF flow patterns, left-handed, right-handed and non-biased, in movement of the beads into the third ventricle from the cerebral aqueduct (aqueduct of Sylvius). In the lateral ventricles, anterior to the third ventricle, CSF flow moved anteriorly along the outer wall of the ventricle to the inner wall and then posteriorly, creating a semicircle. In the cerebral aqueduct, connecting the third and fourth cerebral ventricles, CSF flow moved rostrally in the dorsal region and caudally in the ventral region. Also in the fourth ventricle, clear dorso-ventral differences in fluid flow pattern were observed.
Conclusions:
This is the first visualisation of the orchestrated CSF flow pattern in developing vertebrates using a live animal imaging approach. CSF flow in Xenopus albino larvae showed a largely consistent pattern, with the exception of individual differences in left-right asymmetrical flow in the third ventricle.
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In vitro effects of fetal rat cerebrospinal fluid on viability and neuronal differentiation of PC12 cells
Background:
Fetal cerebrospinal fluid (CSF) contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an in vitro model of neuronal differentiation since they differentiate into sympathetic neuron-like cells in response to growth factors. This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described.
Methods:
In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and beta-III tubulin) was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis.
Results:
PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive.
Conclusions:
Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important in vivo for the promotion of normal brain development. There were significant differences in the effects on PC12 cells compared to primary cortical cells. This suggests there is an interaction in vivo between developmental stage of cells and the composition of CSF. The data presented here support an important, perhaps driving role for CSF composition, specifically neurotrophic factors, in neuronal survival, proliferation and differentiation. The effects of CSF on PC12 cells can thus be used to further investigate the role of CSF in driving development without the confounding issues of using primary cells.
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Phosphorylated tau/amyloid beta 1-42 ratio in ventricular cerebrospinal fluid reflects outcome in idiopathic normal pressure hydrocephalus
Background:
Idiopathic normal pressure hydrocephalus (iNPH) is a potentially reversible cause of dementia and gait disturbance that is typically treated by operative placement of a ventriculoperitoneal shunt. The outcome from shunting is variable, and some evidence suggests that the presence of comorbid Alzheimer's disease (AD) may impact shunt outcome. Evidence also suggests that AD biomarkers in cerebrospinal fluid (CSF) may predict the presence of AD. The aim of this study was to investigate the relationship between the phosphorylated tau/amyloid beta 1-42 (ptau/Aβ1-42) ratio in ventricular CSF and shunt outcome in patients with iNPH.
Methods:
We conducted a prospective trial with a cohort of 39 patients with suspected iNPH. Patients were clinically and psychometrically assessed prior to and approximately 4 months after ventriculoperitoneal shunting. Lumbar and ventricular CSF obtained intraoperatively, and tissue from intraoperative cortical biopsies were analyzed for AD biomarkers. Outcome measures included performance on clinical symptom scales, supplementary gait measures, and standard psychometric tests. We investigated relationships between the ptau/Aβ1-42 ratio in ventricular CSF and cortical AD pathology, initial clinical features, shunt outcome, and lumbar CSF ptau/Aβ1-42 ratios in the patients in our cohort.
Results:
We found that high ptau/Aβ1-42 ratios in ventricular CSF correlated with the presence of cortical AD pathology. At baseline, iNPH patients with ratio values most suggestive of AD presented with better gait performance but poorer cognitive performance. Patients with high ptau/Aβ1-42 ratios also showed a less robust response to shunting on both gait and cognitive measures. Finally, in a subset of 18 patients who also underwent lumbar puncture, ventricular CSF ratios were significantly correlated with lumbar CSF ratios.
Conclusions:
Levels of AD biomarkers in CSF correlate with the presence of cortical AD pathology and predict aspects of clinical presentation in iNPH. Moreover, preliminary evidence suggests that CSF biomarkers of AD may prove useful for stratifying shunt prognosis in patients being evaluated and treated for this condition.
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Expression and localization of claudins-3 and -12 in transformed human brain endothelium
Background:
The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12.FindingshCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture.
Conclusions:
These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.
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Detection of oligoclonal IgG kappa and IgG lambda bands in cerebrospinal fluid and serum with HevyliteTM antibodies. Comparison with the free light chain oligoclonal pattern.
Background:
Oligoclonal IgG bands in cerebrospinal fluid that are absent in serum indicate intrathecal IgG synthesis and are a sensitive marker of CNS inflammatory diseases, in particular multiple sclerosis. It may be of interest to determine whether these bands are predominantly IgGκ or IgGλ.
Methods:
We have used Hevylite™ antibodies and developed a technique for detection of oligoclonal IgGκ and IgGλ bands by means of isoelectric focusing followed by immunoblotting. The same technique was used for oligoclonal free κ and free λ detection. Among several techniques tested, affinity immunoblotting appears to be the most sensitive; it can detect less than 1 ng of IgGκ or IgGλ paraprotein. We compared oligoclonal IgG profiles with those of oligoclonal IgGκ and IgGλ. There was good agreement concerning the presence or absence of intrathecal synthesis. We observed the ratios between oligoclonal IgGκ and IgGλ bands, and they did not always match the ratios between free κ and free λ bands. We were also able to detect antigen-specific CSF-restricted oligoclonal IgGκ and IgGλ bands in neuroborreliosis. It remains to be determined subsequently by a clinically-oriented prospective study, whether predominant IgGκ/IgGλ or free κ/free λ can be observed more frequently in particular diseases with oligoclonal IgG synthesis.DiscussionVery sensitive detection of oligoclonal IgGκ and IgGλ bands in cerebrospinal fluid with Hevylite antibodies is feasible; detection of antigen-specific IgGκ or IgGλ is possible as well. In particular situations, e.g. when difficulties arise in distinguishing between oligoclonal and monoclonal pattern, the test may be of considerable clinical value.
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Evaluation of blood-brain barrier transport and CNS drug metabolism in diseased and control brain after intravenous L-DOPA in a unilateral rat model of Parkinson's disease
Background:
Changes in blood-brain barrier (BBB) functionality have been implicated in Parkinson's disease. This study aimed to investigate BBB transport of L-DOPA transport in conjunction with its intra-brain conversion, in both control and diseased cerebral hemispheres in the unilateral rat rotenone model of Parkinson's disease.
Methods:
In Lewis rats, at 14 days after unilateral infusion of rotenone into the medial forebrain bundle, L-DOPA was administered intravenously (10, 25 or 50 mg/kg). Serial blood samples and brain striatal microdialysates were analysed for L-DOPA, and the dopamine metabolites DOPAC and HVA. Ex-vivo brain tissue was analyzed for changes in tyrosine hydroxylase staining as a biomarker for Parkinson's disease severity. Data were analysed by population pharmacokinetic analysis (NONMEM) to compare BBB transport of L-DOPA in conjunction with the conversion of L-DOPA into DOPAC and HVA, in control and diseased cerebral hemisphere.
Results:
Plasma pharmacokinetics of L-DOPA could be described by a 3-compartmental model. In rotenone responders (71%), no difference in L-DOPA BBB transport was found between diseased and control cerebral hemisphere. However, in the diseased compared with the control side, basal microdialysate levels of DOPAC and HVA were substantially lower, whereas following L-DOPA administration their elimination rates were higher.
Conclusions:
Parkinson's disease-like pathology, indicated by a huge reduction of tyrosine hydroxylase as well as by substantially reduced levels and higher elimination rates of DOPAC and HVA, does not result in changes in BBB transport of L-DOPA. Taking the results of this study and that of previous ones, it can be concluded that changes in BBB functionality are not a specific characteristic of Parkinson's disease, and cannot account for the decreased benefit of L-DOPA at later stages of Parkinson's disease.
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Temporal course of cerebrospinal fluid dynamics and amyloid accumulation in the aging rat brain from three to thirty months
Background:
Amyloid accumulation in the brain parenchyma is a hallmark of Alzheimer's disease (AD) and is seen in normal aging. Alterations in cerebrospinal fluid (CSF) dynamics are also associated with normal aging and AD. This study analyzed CSF volume, production and turnover rate in relation to amyloid-beta peptide (Aβ) accumulation in the aging rat brain.
Methods:
Aging Fischer 344/Brown-Norway hybrid rats at 3, 12, 20, and 30 months were studied. CSF production was measured by ventriculo-cisternal perfusion with blue dextran in artificial CSF; CSF volume by MRI; and CSF turnover rate by dividing the CSF production rate by the volume of the CSF space. Aβ40 and Aβ42 concentrations in the cortex and hippocampus were measured by ELISA.
Results:
There was a significant linear increase in total cranial CSF volume with age: 3-20 months (p < 0.01); 3-30 months (p < 0.001). CSF production rate increased from 3-12 months (p < 0.01) and decreased from 12-30 months (p < 0.05). CSF turnover showed an initial increase from 3 months (9.40 day-1) to 12 months (11.30 day-1) and then a decrease to 20 months (10.23 day-1) and 30 months (6.62 day-1). Aβ40 and Aβ42 concentrations in brain increased from 3-30 months (p < 0.001). Both Aβ42 and Aβ40 concentrations approached a steady state level by 30 months.
Conclusions:
In young rats there is no correlation between CSF turnover and Aβ brain concentrations. After 12 months, CSF turnover decreases as brain Aβ continues to accumulate. This decrease in CSF turnover rate may be one of several clearance pathway alterations that influence age-related accumulation of brain amyloid.
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Intrastriatal convection-enhanced delivery results in widespread perivascular distribution in a pre-clinical model
Background:
Convection-enhanced delivery (CED), a direct method for drug delivery to the brain through intraparenchymal microcatheters, is a promising strategy for intracerebral pharmacological therapy. By establishing a pressure gradient at the tip of the catheter, drugs can be delivered in uniform concentration throughout a large volume of interstitial fluid. However, the variables affecting perivascular distribution of drugs delivered by CED are not fully understood. The aim of this study was to determine whether the perivascular distribution of solutes delivered by CED into the striatum of rats is affected by the molecular weight of the infused agent, by co-infusion of vasodilator, alteration of infusion rates or use of a ramping regime. We also wanted to make a preliminary comparison of the distribution of solutes with that of nanoparticles.
Methods:
We analysed the perivascular distribution of 4, 10, 20, 70, 150 kDa fluorescein-labelled dextran and fluorescent nanoparticles at 10 min and 3 h following CED into rat striatum. We investigated the effect of local vasodilatation, slow infusion rates and ramping on the perivascular distribution of solutes. Co-localisation with perivascular basement membranes and vascular endothelial cells was identified by immunohistochemistry. The uptake of infusates by perivascular macrophages was quantified using stereological methods.
Results:
Widespread perivascular distribution and macrophage uptake of fluorescein-labelled dextran was visible 10 min after cessation of CED irrespective of molecular weight. However, a significantly higher proportion of perivascular macrophages had taken up 4, 10 and 20 kDa fluorescein-labelled dextran than 150 kDa dextran (p < 0.05, ANOVA). Co-infusion with vasodilator, slow infusion rates and use of a ramping regime did not alter the perivascular distribution. CED of fluorescent nanoparticles indicated that particles co-localise with perivascular basement membranes throughout the striatum but, unlike soluble dextrans, are not taken up by perivascular macrophages after 3 h.
Conclusions:
This study suggests that widespread perivascular distribution and interaction with perivascular macrophages is likely to be an inevitable consequence of CED of solutes. The potential consequences of perivascular distribution of therapeutic agents, and in particular cytotoxic chemotherapies, delivered by CED must be carefully considered to ensure safe and effective translation to clinical trials.
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