A simple novel approach for detecting blood-brain barrier permeability using GPCR internalization.

AIMS: Impairment of blood-brain barrier (BBB) is involved in numerous neurological diseases from developmental to aging stages. Reliable imaging of increased BBB permeability is therefore crucial for basic research and preclinical studies. Today, the analysis of extravasation of exogenous dyes is the principal method to study BBB leakage. However, these procedures are challenging to apply in pups and embryos and may appear difficult to interpret. Here we introduce a novel approach based on agonist-induced internalization of a neuronal G protein-coupled receptor widely distributed in the mammalian brain, the somatostatin receptor type 2 (SST2). METHODS: The clinically approved SST2 agonist octreotide (1 kDa), when injected intraperitoneally does not cross an intact BBB. At sites of BBB permeability, however, OCT extravasates and induces SST2 internalization from the neuronal membrane into perinuclear compartments. This allows an unambiguous localization of increased BBB permeability by classical immunohistochemical procedures using specific antibodies against the receptor. RESULTS: We first validated our approach in sensory circumventricular organs which display permissive vascular permeability. Through SST2 internalization, we next monitored BBB opening induced by magnetic resonance imaging-guided focused ultrasound in murine cerebral cortex. Finally, we proved that after intraperitoneal agonist injection in pregnant mice, SST2 receptor internalization permits analysis of BBB integrity in embryos during brain development. CONCLUSIONS: This approach provides an alternative and simple manner to assess BBB dysfunction and development in different physiological and pathological conditions.

Holographic laser Doppler imaging of microvascular blood flow.

We report on local superficial blood flow monitoring in biological tissue from laser Doppler holographic imaging. In time-averaging recording conditions, holography acts as a narrowband bandpass filter, which, combined with a frequency-shifted reference beam, permits frequency-selective imaging in the radio frequency range. These Doppler images are acquired with an off-axis Mach-Zehnder interferometer. Microvascular hemodynamic components mapping is performed in the cerebral cortex of the mouse and the eye fundus of the rat with near-infrared laser light without any exogenous marker. These measures are made from a basic inverse-method analysis of local first-order optical fluctuation spectra at low radio frequencies, from 0 Hz to 100 kHz. Local quadratic velocity is derived from Doppler broadenings induced by fluid flows, with elementary diffusing wave spectroscopy formalism in backscattering configuration. We demonstrate quadratic mean velocity assessment in the 0.1-10 mm/s range in vitro and imaging of superficial blood perfusion with a spatial resolution of about 10 micrometers in rodent models of cortical and retinal blood flow.

New pool of cortical interneuron precursors in the early postnatal dorsal white matter.

The migration of cortical γ-aminobutyric acidergic interneurons has been extensively studied in rodent embryos, whereas few studies have documented their postnatal migration. Combining in vivo analysis together with time-lapse imaging on cortical slices, we explored the origin and migration of cortical interneurons during the first weeks of postnatal life. Strikingly, we observed that a large pool of GAD65-GFP-positive cells accumulate in the dorsal white matter region during the first postnatal week. Part of these cells divides and expresses the transcription factor paired box 6 indicating the presence of local transient amplifying precursors. The vast majority of these cells are immature interneurons expressing the neuronal marker doublecortin and partly the calcium-binding protein calretinin. Time-lapse imaging reveals that GAD65-GFP-positive neurons migrate from the white matter pool into the overlying anterior cingulate cortex (aCC). Some interneurons in the postnatal aCC express the same immature neuronal markers suggesting ongoing migration of calretinin-positive interneurons. Finally, bromodeoxyuridine incorporation experiments confirm that a small fraction of interneurons located in the aCC are generated during the early postnatal period. These results altogether reveal that at postnatal ages, the dorsal white matter contains a pool of interneuron precursors that divide and migrate into the aCC.

Lack of barrels in the somatosensory cortex of monoamine oxidase A-deficient mice: role of a serotonin excess during the critical period.

In a transgenic mouse line (Tg8) deficient for the gene encoding monoamine oxidase A (MAOA), we show that the primary somatosensory cortex (S1) lacks the characteristic barrel-like clustering of layer IV neurons, whereas normal pattern formation exists in the thalamus and the trigeminal nuclei. No barrel-like patterns were visible with tenascin or serotonin immunostaining or with labeling of thalamocortical axons. An excess of brain serotonin during the critical period of barrel formation appears to have a causal role in these cortical abnormalities, since early administration of parachlorophenylalanine, an inhibitor of serotonin synthesis, in Tg8 pups restored the formation of barrels in S1, whereas inhibition of catecholamine synthesis did not. Transient inactivation of MAOA in normal newborns reproduced a barrelless phenotype in parts of S1.

Integrating whole transcriptome assays on a lab-on-a-chip for single cell gene profiling.

To correlate gene expression profiles to fundamental biological processes such as cell growth, differentiation and migration, it is essential to work at the single cell level. Gene expression analysis always starts with the relatively low efficient reverse transcription (RT) of RNA into complementary DNA (cDNA), an essential step as unprocessed RNAs will not be analysed further. In this paper, we present a novel method for RT that uses microfluidics to manipulate nanolitre volumes. We compare our method to conventional protocols performed in microlitre volumes. More specifically, reverse transcription was performed either in a polydimethylsiloxane (PDMS) rotary mixer or in a tube, using a single cell amount of mouse brain RNA (10 pg), and was followed by a template-switching PCR (TS-PCR) amplification step. We demonstrate that, using the microfluidic protocol, 74% of the genes expressed in mouse brain were detected, while only 4% were found with the conventional approach. We next profiled single neuronal progenitors. Using our microfluidic approach, i.e. performing cell capture, lysis and reverse transcription on-chip followed by TS-PCR amplification in tube, a mean of 5000 genes were detected in each neuron, which corresponds to the expected number of genes expressed in a single cell. This demonstrates the outstanding sensitivity of the microfluidic method.

PLC-beta1, activated via mGluRs, mediates activity-dependent differentiation in cerebral cortex.

During development of the cerebral cortex, the invasion of thalamic axons and subsequent differentiation of cortical neurons are tightly coordinated. Here we provide evidence that glutamate neurotransmission triggers a critical signaling mechanism involving the activation of phospholipase C-beta1 (PLC-beta1) by metabotropic glutamate receptors (mGluRs). Homozygous null mutation of either PLC-beta1 or mGluR5 dramatically disrupts the cytoarchitectural differentiation of 'barrels' in the mouse somatosensory cortex, despite segregation in the pattern of thalamic innervation. Furthermore, group 1 mGluR-stimulated phosphoinositide hydrolysis is dramatically reduced in PLC-beta1-/- mice during barrel development. Our data indicate that PLC-beta1 activation via mGluR5 is critical for the coordinated development of the neocortex, and that presynaptic and postsynaptic components of cortical differentiation can be genetically dissociated.

The transcription factor Pax6 is required for development of the diencephalic dorsal midline secretory radial glia that form the subcommissural organ.

During brain development, Pax6 is expressed in specific regions of the diencephalon including secretory cells of the subcommissural organ (SCO), a circumventricular organ at the forebrain-midbrain boundary that originates from the pretectal dorsal midline neuroepithelial cells beneath the posterior commissure (PC). Homozygous small eye (Sey/Sey) mice lack functional Pax6 protein and fail to develop the SCO, a normal PC and the pineal gland. Small eye heterozygotes (Sey/+) show defective development of the SCO's basal processes which normally penetrate the PC, indicating that normal development of the gland requires normal Pax6 gene-dosage. A correlation between the defects of SCO formation and altered R- and OB-cadherin expression patterns in the SCO is observed in mutants suggesting a role for cadherins in SCO development.

Effects of monoamine oxidase A inhibition on barrel formation in the mouse somatosensory cortex: determination of a sensitive developmental period.

Genetic inactivation of monoamine oxidase A (MAOA) in C3H/HeJ mice causes a complete absence of barrels in the somatosensory cortex, and similar alterations are caused by pharmacological inhibition of MAOA in wild type mice. To determine when and how MAOA inhibition affects the development of the barrel field, the MAOA inhibitor clorgyline was administered to mice of the outbred strain OF1 for various time periods between embryonic day 15 (E15) and postnatal day 7 (P7), and the barrel fields were analyzed with cytochrome oxidase and Nissl stains in P10 and adult mice. High-pressure liquid chromatography measures of brain serotonin (5-HT) showed three- to eightfold increases during the periods of clorgyline administration. Perinatal mortality was increased and weight gain was slowed between P3 and P6. Clorgyline treatments from E15 to P7 or from P0 to P7 disrupted the formation of barrels in the anterior snout representation and in parts of the posteromedial barrel subfield (PMBSF). Treatments from P0 to P4 caused similar although less severe barrel field alterations. Clorgyline treatments only during embryonic life or starting on P4 caused no detectable abnormalities. In cases with barrel field alterations, a rostral-to-caudal gradient of changes was noted: Rostral barrels of the PMBSF were most frequently fused and displayed an increased size tangentially. Thus, MAOA inhibition resulting in increased brain levels of 5-HT affects barrel development during the entire first postnatal week, with a sensitive period between P0 and P4. The rostral-to-caudal gradient of changes in the barrel field parallels known developmental gradients in the sensory periphery and in the maturation thalamocortical afferents. The observed barrel fusions could correspond to a default in the initial segregation of thalamic fibers or to a continued, exuberant growth of these fibers that overrides the tangential domain that is normally devoted to individual whiskers.

Transmission blocks: clinical and biological rationales.

This paper describes the clinical and biological rationales for the use of transmission blocks. Clinical advantages over the use of full-thickness blocks applied part way through the course of therapy include the use of only one set of fields, blocks, and beam calculations, and less complex chart recording. There is a net saving in time required for the preparation and treatment of the patient. There is also a quality assurance advantage since the impact of a potential error in block positioning is reduced. In terms of biological advantages, it is demonstrated that the linear-quadratic iso-effect model can be applied to predict an improvement of up to 10% in the therapeutic ratio if transmission blocks are used instead of full-thickness blocks.

Effects of genetic depletion of monoamines on somatosensory cortical development.

Raised levels of serotonin cause alterations in the development of the barrelfield of the primary somatosensory cortex (S1) in rodents. We examined the development of S1 in genetic mouse models in which the levels of serotonin and/or dopamine and noradrenaline are drastically reduced. Mice lacking the vesicular monoamine transporter type 2 (VMAT2 KO) are hypomorphic with rare pups surviving until postnatal day (P) 6. Serotonin, dopamine and noradrenaline are almost undetectable in the brain. In S1 we find that the segregation of thalamocortical axons into whisker patterns is delayed by 1 day and that layer IV granular neurons fail to form normal barrels. Moreover, the growth of cortical layers II-IV is reduced. Despite severe malnutrition, we show that these alterations are not caused by increased cell death in the thalamus or S1. Moreover, the maturation of cortical neurons is not altered as reflected by calcium-binding protein immunolabeling. Mice lacking both VMAT2 and monoamine oxidase type A (MAOA) were generated. VMAT2-MAOA DKO mice are hypomorphic but survive until P13. Increased levels of serotonin but profoundly reduced dopamine and noradrenaline levels are found in the brains. In S1, alterations are similar to those observed in MAOA KO mice: thalamocortical axons and granular neurons failed to form barrels. In addition there is a severe reduction in the thickness of the upper cortical layers as in the VMAT2 KO mice. These results show that monoamines have no instructive effect per se on the formation of thalamocortical patterning in S1. However, monoamines appear to be essential for the normal cytoarchitectonic maturation of the granular (IV) and supragranular cortical layers (II-III). Since developmental cell death and chemoarchitectonic differentiation of these neurons are not modified, it is possible that these alterations result from migration defects and/or from altered synaptic maturation.

Practical aspects of transmission cord blocks in radiotherapy.

We present the advantages of using partial transmission cord blocks throughout treatment, as opposed to adding full-thickness blocks near the end. Such blocks reduce the risk of block omission or mispositioning and require less total time for construction. We also present an argument for the existence of an optimal width for cord blocks used in mediastinal treatments. A figure of merit has been derived which quantifies the tradeoff between narrow blocks, which increase the variation in dose across the block shadow and the risk of positioning errors, and wide blocks, which may unnecessarily shield potential tumor sites. For 60Co, 4- and 10-MV beams, the figure of merit peaks at block widths of 2.0-2.5 cm at the level of the cord. Effective transmission data for cord blocks constructed of cerrobend are given for those three beams. Quality assurance studies show that transmission through cerrobend blocks can be controlled to the required precision.

Usefulness of bronchoalveolar lavage for diagnosis of acute and persistent respiratory syncytial virus lung infections in guinea pigs.

To investigate whether bronchoalveolar lavage (BAL) fluid specimens can be used to diagnose acute and persistent respiratory syncytial virus (RSV) lung infections in guinea pigs, we tested BAL fluid and lung tissue specimens for evidence of viral infection, and compared BAL cytology between infected and uninfected animals. RSV-inoculated guinea pigs were studied during acute bronchiolitis (days 3 and 7 postinoculation), convalescence (Day 14 postinoculation), and persistent infection (Days 28 and 60 postinoculation), and were compared to the sham-infected control animals. BAL and lung tissue specimens were cultured for virus and tested by immunocytochemistry for viral protein. A reverse transcription-polymerase chain reaction (RT-PCR) method was used to test for viral nucleic acid. Total and differential BAL cell counts were compared between RSV-inoculated and control animals on each study day. In BAL specimens, replicating RSV was isolated by culture in one out of four of the animals on Day 3 postinoculation; immunocytochemistry for RSV antigens was positive in all virus-exposed animals from Days 3-14 postinoculation, and viral nucleic acid was detected by RT-PCR in one-fourth of the animals on Day 3 postinoculation. In contrast, replicating virus, viral antigens, and viral nucleic acid were documented in lung tissues obtained from the same RSV-infected animals on all study days. BAL specimens of RSV-inoculated animals contained more eosinophils on all study days (two-tailed P value < 0.01) compared to the controls. The results of this animal study demonstrate that BAL fluid is not useful for diagnosis of persistent RSV infection. However, BAL fluid may be helpful for the documentation of acute RSV lung infection when immunocytochemistry may provide a more accurate test for virus detection than RT-PCR or viral culture.

High-speed wave-mixing laser Doppler imaging in vivo.

An interferometric method for parallel optical spectroscopy in the kilohertz range is reported, as well as its experimental validation in the context of high-speed laser Doppler imaging in vivo. The interferometric approach enables imaging in the low light conditions of a 2 kHz frame rate recording with a complementary metal-oxide semiconductor camera. Observation of mice craniums with near-infrared (lambda=785 nm) laser light in reflection configuration is reported. Doppler spectral images allegedly sensitive to blood flow are sequentially measured at several optical frequency detunings, to shift the spectral range of analysis in the radio-frequency spectrum.

Frequency-domain wide-field laser Doppler in vivo imaging.

We present a new instrument, based on a low-frame-rate (8 Hz) CCD camera used in a heterodyne optical-mixing configuration, that can create wide-field laser Doppler maps. As an illustration, we show results obtained in a mouse brain, in vivo, showing the Doppler signature of blood flow. The instrument is based on a frequency-shifting digital holography scheme.

Pulmonary and cutaneous oxygen uptake and oxygen consumption of isolated skin in the frog, Rana pipiens.

Oxygen uptake and consumption rates were measured in intact and isolated skin from the amphibian Rana pipiens to determine the percentage of total cutaneous O2 uptake that is consumed by the skin itself. Paired measurements, using open respirometry, were done on intact and isolated skin. In normoxic water oxygen uptake across the cutaneous surface was always greater than O2 consumed by isolated skin. Closed respirometry was used to determine the effect of declining water PO2 on cutaneous uptake across intact skin and oxygen consumption by the isolated skin. Both intact skin uptake and isolated skin consumption were related significantly to water PO2 (150-100 mm Hg). Assuming O2 consumption of the isolated skin to be same as in situ, calculations showed that when water PO2 was high (150 mm Hg), about 40% of total cutaneous O2 uptake was consumed by the skin. At low PO2 (100 mm Hg) this figure was about 20%. Thus in the face of declining water PO2, a greater percentage of total cutaneous uptake goes to satisfy the oxygen requirements of other tissues.

Persistence of respiratory syncytial virus (RSV) infection and development of RSV-specific IgG1 response in a guinea-pig model of acute bronchiolitis.

Acute respiratory syncytial virus (RSV) bronchiolitis in children can result in sequelae of recurrent wheezing and asthma and production of RSV-specific immunoglobulin E (IgE), but the pathogenesis of these sequeleae is poorly understood. Guinea-pigs experimentally inoculated with human RSV show histological evidence of acute bronchiolitis and chronic persistence of viral antigens and genome in the lungs; whether this persistence is due to infectious replicating virus, and whether infected animals develop RSV-specific immunoglobulin G1 (IgG1) (the main class of antibody involved in guinea-pig allergic responses) is unknown. Guinea-pigs were inoculated intranasally with human RSV or with uninfected cell culture supernatant. At times ranging 1-60 days postinoculation, the viral titre in the lung was determined by immunoplaque assay (a method combining viral culture and immunocytochemistry). Serum titres of RSV-specific IgG1 antibodies were determined by enzyme-linked immunosorbent assay. Bronchiolar inflammation was assessed on coded lung sections, by using a semiquantitative, histological scoring system based on features of human acute bronchiolitis. Infectious RSV was cultured from the lungs of infected animals on all study days, with maximal viral replication observed on Day 3. RSV-specific IgG1 antibodies were detected in all RSV-inoculated animals from Day 7 onward, with the highest antibody titre measured on Day 28. RSV-inoculated guinea-pigs had maximal bronchiolar inflammation on Day 7, and had significantly increased polymorphonuclear cell infiltrates on Days 28 and 60. Respiratory syncytial virus chronically persists as infectious virus in the guinea-pig lung. Infected animals develop an anti-respiratory syncytial virus immunoglobulin G1 antibody response, histological evidence of acute bronchiolitis, and chronic airway inflammation. Persistent respiratory syncytial virus lung infection may be important in the pathogenesis of postbronchiolitis wheezing and asthma in children.

Defect of tyrosine hydroxylase-immunoreactive neurons in the brains of mice lacking the transcription factor Pax6.

In the CNS, the lack of the transcription factor Pax6 has been associated with early defects in cell proliferation, cell specification, and axonal pathfinding of discrete neuronal populations. In this study, we show that Pax6 is expressed in discrete catecholaminergic neuronal populations of the developing ventral thalamus, hypothalamus, and telencephalon. In mice lacking Pax6, these catecholaminergic populations develop abnormally: those in the telencephalon are reduced in cell number or absent, whereas those in the ventral thalamus and hypothalamus are greatly displaced and densely packed. Catecholaminergic neurons of the substantia nigra (SN) and the ventral tegmental area (VTA) do not express Pax6 protein. Nevertheless, mice lacking Pax6 display an altered pathfinding of SN-VTA projections: instead of following the route of the medial forebrain bundle ventrally, most of the SN-VTA projections are deflected dorsorostrally at the pretectal-dorsal thalamic transition zone and in the dorsal thalamic alar plate. Moreover, some catecholaminergic neurons are displaced dorsally to an ectopic location at the pretectal-dorsal thalamic transition zone. Interestingly, from the pretectal-dorsal thalamic to the dorsal thalamic-ventral thalamic transition zones, mice lacking Pax6 display an ectopic ventral to dorsal expansion of the chemorepellant/chemoattractive molecule, Netrin-1. This may be responsible for both the altered pathway of catecholaminergic fibers and the ectopic location of catecholaminergic neurons in this region.

A model of latent adenovirus 5 infection in the guinea pig (Cavia porcellus).

A model of adenovirus 5 (Ad5) infection was developed in guinea pigs to begin to study its role in the pathogenesis of peripheral lung inflammation. Forty animals were inoculated intranasally with 10(7.0) pfu of Ad5/animal, and 15 animals inoculated with sterile culture media served as controls. Viral titres were 10(4.4), 10(6.1), 10(5.2), and 10(2.9) pfu/animal, on days 1, 3, 4, and 7 after infection, respectively. In situ hybridization to viral DNA and immunocytochemistry for Ad5 E1A protein localized the virus to airway and alveolar epithelial cells. Histologic examination showed an extensive inflammatory cell infiltration around the airways, with epithelial necrosis and an alveolar exudate that caused localized alveolar collapse in the infected areas. Immunocytochemistry identified the cells in the infiltrate as cytotoxic T cells. Although all animals 20 and 47 days after infection had seroconverted to Ad5, virus was not detected in these groups either by viral plaque assay or in situ hybridization. Ad5 E1A DNA was detected by polymerase chain reaction in five of six animals 20 days after infection and in five of five animals 47 days after infection. In these same animals, E1A protein was detected 20 days after infection in two and 47 days after infection in one while persistent bronchiolitis was observed in four and three animals 20 and 47 days after infection, respectively. These results demonstrate that the guinea pig provides a useful model to study the role of Ad5 infection in chronic airway inflammation.

The effect of latent adenovirus 5 infection on cigarette smoke-induced lung inflammation.

The aim of this study was to test the hypothesis that latent adenovirus (Ad) 5 infection increases the lung inflammation that follows a single acute exposure to cigarette smoke. A recently developed model of latent adenoviral infection in guinea-pigs was used. Twelve animals were infected with Ad5 (10(8) plaque-forming units) and 12 animals were sham-infected. Thirty five days later six Ad5-infected and six sham-infected animals were exposed to the smoke from five cigarettes. The remaining animals were used as controls for both infection and smoking. As markers of inflammation, the volume fraction of macrophages, T-lymphocytes, neutrophils and eosinophils were measured by quantitative histology. We found that latent Ad5-infection alone, doubled the number of macrophages in the lung parenchyma and that smoking alone, doubled the volume fraction of neutrophils in the airway wall and the volume fraction of macrophages in the lung parenchyma. Neither viral infection nor smoking, alone, had an effect on T-lymphocytes or eosinophils. However, the combination of viral infection and smoking doubled the T-lymphocyte helper cells and quadrupled the volume fraction of macrophages in the lung parenchyma. We conclude that in guinea-pigs, latent adenovirus 5 infection increases the inflammation that follows a single acute exposure to cigarette smoke, by increasing the volume fraction of macrophages and T-lymphocyte helper cells.

Effects of respiratory syncytial virus persistence on airway responsiveness and inflammation in guinea-pigs.

Recurrent wheezing and asthma often develop after acute respiratory syncytial virus (RSV) bronchiolitis, but the mechanisms of these sequelae are poorly understood. Using a guinea-pig model of human RSV lung infection, the effects of long-term viral persistence on three hallmarks of asthma: nonspecific airway responsiveness, airway inflammation and airway remodelling were examined. Guinea-pigs were studied 100 days after intranasal instillation of either human RSV or uninfected vehicle, using: 1) acetylcholine challenge to test for airway hyperresponsiveness (AHR); 2) lung histology to quantify the numbers of airway eosinophils and metachromatic cells (mast cells/basophils); 3) airway morphometry of the areas of the airway subepithelial connective tissue, smooth muscle and adventitia, to test for airway remodelling; and 4) immunohistochemistry to identify lung cells containing RSV antigens. The RSV-inoculated group had significantly elevated AHR and airway eosinophils compared to uninfected control animals (p<0.05). There were no significant differences between the two groups in terms of numbers of airway metachromatic cells, or the areas of subepithelial connective tissue, smooth muscle or adventitia. Viral proteins were identified by immunohistochemistry within several types of lung cells. In conclusion, long-term persistence of respiratory syncytial virus in the guinea-pig lung is associated with airway hyperresponsiveness and airway eosinophilia, and these changes may be pertinent to the pathogenesis of postbronchiolitis wheezing and asthma in children.