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No description available.

Breakdown of the disc extracellular matrix is thought to arise from increased activity of matrix metalloproteinases (MMPs). Aggrecan, one of the major disc matrix macromolecules, is degraded through action of MMPs and aggrecanases and its concentration falls early in the degeneration process. Loss of the constituent glycosaminoglycans (GAGs), which are osmotically active, leads to a decrease in osmotic pressure and subsequently to a fall in tissue hydration. Apart from the major biomechanical consequences, fall in extracellular osmolarity is known to influence cell function. Apart from aggrecan, other macromolecules such as fibronectin are also broken down by MMPs. Fibronectin fragments (Fnfs) have been identified in degenerate discs. Such Fnfs have been found to stimulate production and activity of MMPs in articular cartilage but little is known about the effect of these fragments on disc cells. The aim of the work was thus to determine whether Fnf stimulated expression of MMPs and hence induced aggrecan breakdown and loss in the disc and whether extracellular osmolarity influenced this potential response. NP cells or explants were harvested from adult bovine caudal discs. They were cultured in DMEM culture medium over a range of osmolarities with or without Fnf treatment. Profiles of gene expression of MMPs and their inhibitors and effect of changes in osmolarity on expression of selected MMPs were determined. The effect of Fnf on responses of cells and tissue explants from the central region of the disc, the nucleus pulposus (NP) and the role of changes in extracellular osmolarity in relation to GAG loss and expression of selected MMPs was then examined both at the protein level and by gene profiling using a microarray. My results showed that expression of MMPs by disc cells is regulated by extracellular osmolarity rather than the 30 kD Fnf, with the level of some MMPs secreted by disc cells and involved in degradation of disc matrix rising as osmolarity falls. These results could explain in part the finding that MMP expression increases with degree of disc degeneration i.e. with loss of aggrecan and fall in extracellular osmolarity. These also suggested that a fall in osmolarity could induce a degenerative cascade with proteolytic digestion of aggrecan leading to a fall in osmolarity and hence a further increase in proteinase expression and matrix degradation.

Dry eye is a complex, multi-factorial disease that results in a compromised tear film and ocular surface. Clinicians and researchers alike have historically relied on an individual’s symptoms to diagnose and manage the condition, due to a lack of reliable objective methods for quantifying disease presence and severity. Of late, parameters such as tear film osmolarity and tear meniscus height have shown promise as valid methods for enumerating characteristics of the tear film that may aid the diagnosis of dry eye. Two new technologies have recently been introduced that can measure said parameters. The TearLab™ is a novel handheld nano-osmometer capable of measuring tear film osmolarity on samples as small as 50 nL. The device uses electrical conductance to measure osmolarity, and the small sample requirements purportedly allows the device to minimally disturb the natural state of the tear film. The RTVue-100 is a spectral-, or Fourier-domain optical coherence tomographer that has the ability to generate high resolution, cross-sectional images of the tear meniscus, and subsequently measure tear meniscus height. As little is published on the use of these technologies to evaluate the tear film, a series of studies was completed to determine their performance in both a normal and dry eye population.

Dry eye is a complex, multi-factorial disease that results in a compromised tear film and ocular surface. Clinicians and researchers alike have historically relied on an individual’s symptoms to diagnose and manage the condition, due to a lack of reliable objective methods for quantifying disease presence and severity. Of late, parameters such as tear film osmolarity and tear meniscus height have shown promise as valid methods for enumerating characteristics of the tear film that may aid the diagnosis of dry eye. Two new technologies have recently been introduced that can measure said parameters. The TearLab™ is a novel handheld nano-osmometer capable of measuring tear film osmolarity on samples as small as 50 nL. The device uses electrical conductance to measure osmolarity, and the small sample requirements purportedly allows the device to minimally disturb the natural state of the tear film. The RTVue-100 is a spectral-, or Fourier-domain optical coherence tomographer that has the ability to generate high resolution, cross-sectional images of the tear meniscus, and subsequently measure tear meniscus height. As little is published on the use of these technologies to evaluate the tear film, a series of studies was completed to determine their performance in both a normal and dry eye population.

Chez les vertébrés, les neurones qui contactent le LCR (NcLCR) sont présents autour des cavités ventriculaires et tout le long du canal central (cc). Par la combinaison d'enregistrements électrophysiologiques sur tranche de tronc cérébral et d'analyses immunohistochimiques, nous avons réalisé la première caractérisation de cette population neuronale chez la souris adulte. Nous montrons que les NcLCR sont présents autour du cc au niveau du complexe vagal dorsal (CVD), une structure bulbaire impliquée dans la régulation des fonctions autonomes, où ils sont principalement GABAergiques, reçoivent des afférences synaptiques GABA/Glycinergiques et expriment le canal PKD2L1 ("polycystin kidney disease 2-like 1"), un membre de la famille des canaux TRP ("transient receptor potential"). Nous montrons que l'activité de PKD2L1 est modulée par les variations de pH et d'osmolarité et que son augmentation module l'excitabilité des NcLCR. Finalement, nous démontrons que les NcLCR existent dans un état de maturité intermédiaire caractérisé par propriétés fonctionnelles de neurones matures combinées à la conservation de l'expression de marqueurs d'immaturités.Les NcLCR étant stratégiquement positionné entre le LCR et le parenchyme, ils pourraient détecter des signaux circulant grâce à l'activation de PKD2L1 puis distribuer le message collecté à leurs partenaires. Un tel rôle, apparaît particulièrement intéressant au niveau du CVD, un site de régulation majeur des fonctions autonomes et pourrait être démontré par l'identification du réseau neuronal où les NcLCR sont intégrés. / In vertebrates, cerebrospinal fluid contacting neurons (CSF-cN) are present around the ventricular cavities and along the central canal (cc). In this study, by the combination of whole cell patch-clamp recordings on brainstem slice and immunohistochemistry analysis, we realize the first characterization of this neuronal population in adult mice. We show that CSF-cN are present around the cc in the dorsal vagal complex (DVC), a major hindbrain structure regulating autonomic functions. There, CSF-cN are mostly GABAergics, receive GABA- and glycinergic synaptic entries and express functional polycystin kidney disease 2-like 1 (PKD2L1) channels. These channels are a subtype of the transient receptor potential (TRP) channels superfamily and this study represent the first analysis of PKD2L1 properties in a native system. We show that PKD2L1 channel activity is modulated by variations in extracellular pH and osmolarity and in turn, an enhanced activity of only few PKD2L1 channels participates in the modulation of CSF-cN excitability. Finally, we demonstrate that CSFcN exhibit another interesting property since they exist in an intermediate stage of maturity by displaying many mature functional properties combined to the conservation of the expression of immature markers.Because CSF-cN are strategically positioned between CSF and parenchyma, they could detect circulating signals through PKD2L1 activation and convey the collected messages to cellular partners. Such a role might be particularly relevant at the level of the DVC a major regulatory site for autonomic functions and should be demonstrated by identifying and characterizing the neuronal network CSFcN are involved in.

A nefropatia induzida por contraste (NIC) é uma nefropatia aguda, secundária à administração intravascular de meios de contraste iodado (MCI), cujas propriedades físico-químicas, especialmente a osmolaridade, relacionam-se ao seu desenvolvimento. Dentre os mecanismos fisiopatológicos desta enfermidade destacam-se a vasoconstrição intrarrenal prolongada, consequente redução da perfusão renal, hipóxia e isquemia medulares, associada ao dano tubular renal devido à citotoxicidade do contraste. Frente à existência de poucas informações relacionadas a estes mecanismos na literatura médico-veterinária e a grande demanda de exames de tomografia computadorizada que utilizam contrastes, objetivaram-se caracterizar e comparar os efeitos renais da administração intravenosa de MCI não iônicos de diferentes osmolaridades, em grupos de cães com fatores de risco para o desenvolvimento da NIC, por meio das avaliações ultrassonográficas modo B, Doppler colorido, de amplitude e pulsado, pareada aos exames laboratoriais, a fim de estimar indiretamente o potencial nefrotóxico de cada contraste. Este estudo também objetivou verificar a ocorrência da NIC. Constituíram-se dois grupos de acordo com o MCI utilizado: o grupo GIH (11 cães receberam iohexol [baixa osmolaridade]) e o grupo GID (sete cães receberam iodixanol [isosmolar]). Administrou-se a dose de 600 mgI/kg/IV em ambos. Avaliaram-se os seguintes aspectos renais antes da administração do MCI (momento basal) e após 1,5 horas, 24 horas e 48 horas: morfometria (comprimento e volume), morfologia, ecogenicidade cortical e perfusão renais e resistência vascular intrarrenal (índices hemodinâmicos de resistividade e pulsatilidade), realizou-se ainda exame de urina I e se mensuraram as razões gama-glutamil transferase:creatinina (GGT:C) e proteína:creatinina (RPC) urinárias e a concentração sérica de creatinina. Estipulou-se avaliar os aspectos sonográficos do rim esquerdo, desde que ambos os rins apresentassem características morfométricas, morfológicas, de ecogenicidade cortical e perfusão, semelhantes na abordagem inicial de cada paciente. Os grupos apresentaram comportamentos similares para comprimento, RPC, exame de urina I e creatinina sérica. Constataram-se aumentos significativos do índice de resistividade (IR) e da razão GGT:C urinária e evidências de aumento significativo do volume renal, 1,5 horas após a administração do contraste, somente no grupo que recebeu iohexol. Em relação ao índice de pulsatilidade, embora ambos os grupos tenham apresentado comportamentos não similares, não se detectaram diferenças significativas entre o momento basal e os demais. Concluiu-se que o IR foi capaz de monitorar a hemodinâmica intrarrenal e demonstrar, assim como a razão GGT:C urinária, maior potencial nefrotóxico do iohexol, quando comparado ao iodixanol. Dessa forma, considerou-se o iodixanol uma opção favorável para cães com fatores de risco para o desenvolvimento da NIC. Um cão de 14 anos de idade, portador de insuficiência cardíaca e disfunção renal preexistente, desenvolveu a forma subclínica da NIC, reconhecida pela elevação de 0,5 mg/dL na creatinina sérica basal, 24 horas após a administração intravenosa do iohexol. A redução da perfusão renal verificada 1,5 horas após a utilização do contraste pode ser preditiva de NIC. De modo semelhante ao observado no homem, idade avançada, insuficiência cardíaca e disfunção renal preexistente, principalmente quando associadas, constituem fatores de risco para o desenvolvimento da NIC em cães / Contrast-induced nephropathy (CIN) is a type of acute nephropathy, secondary to intravascular administration of iodinated contrast media (ICM). The physicochemical properties of ICM, particularly the osmolarity of the media, are related to the development of CIN. The most important mechanisms of this nephropathy are intrarenal prolonged vasoconstriction, medular hypoxia, and ischemia associated with renal tubular damage due to contrast cytotoxicity. Owing to the limited information available in veterinary literature regarding these mechanisms and increase in the number of contrast-enhanced computed tomography examinations performed, this study aims to characterize and compare the renal effects of intravenous administration of two nonionic ICM of different osmolarities in groups of dogs with risk factors for CIN development, by using a B-mode, color, power- and pulsed-wave Doppler ultrasonography, and other laboratory tests, in order to indirectly estimate the nephrotoxic potential of each contrast. This study also aims to investigate the occurrence of CIN. The following two groups were established according to the nonionic ICM used: the GIH group (11 dogs administered iohexol [low osmolarity]) and the GID group (seven dogs administered iodixanol [iso-osmolarity]). Both the groups were administered the same dose (600 mgI/kg/IV). The following renal aspects were evaluated before administration of ICM (baseline) and after 1.5 h, 24 h, and 48 h: renal morphometry (length and volume), renal morphology, cortical echogenicity, renal perfusion, and intrarenal vascular resistance (resistive and pulsatility indices); in addition, urinalysis was performed, and urinary gamma-glutamyl transferase:creatinine ratio (GGT:C), urinary protein:creatinine ratio (UPC), and serum creatinine were also measured. The sonographic aspects of the left kidney were evaluated, only if both kidneys presented similar morphometry, morphology, cortical echogenicity, and perfusion during the first assessment of each patient. Both groups showed similar characteristics with respect to the length, UPC ratio, urinalysis, and serum creatinine levels. Significant increases were observed in the resistive index (RI) and urinary GGT:C, and evidence of significant increase was observed in the renal volume only in the GIH group, 1.5 h after contrast administration. No similarity was observed with respect to the pulsatility index in both the groups; however, there were no significant differences between baseline and 1.5-, 24- and 48-h time points. In conclusion, RI can be used to monitor intrarenal hemodynamics, and along with the urinary GGT:C, revealed that iohexol had higher nephrotoxic potential than iodixanol. Thus, iodixanol was considered a favorable option for dogs with risk factors for CIN development. A 14-year-old dog with heart failure and pre-existing renal dysfunction developed subclinical CIN, due to an increase in the serum creatinine (0.5 mg/dL) 24 h after intravenous iohexol administration. The decrease in renal perfusion observed 1.5 h after ICM administration may be predictive of CIN. As observed in humans, advanced age, heart failure, and pre-existing renal dysfunction, especially various associated factors, should be considered as risk factors for the development of CIN in dogs

Egg activation is a series of highly coordinated processes that prepare the mature oocyte for embryogenesis. Typically associated with fertilisation, egg activation results in the resumption of the cell cycle, expression of maternal mRNAs and cross-linking of the vitelline membrane. While some aspects of egg activation, such as initiation factors in mammals and environmental cues in sea animals, have been well-documented, the mechanics of egg activation in many animals are still not well understood. This is especially true for animals where fertilisation and egg activation are unlinked. In order to elucidate how egg activation is regulated independently of fertilisation, I use Drosophila melanogaster as a model system. This insect provides extensive genetic tools, ease of manipulation for experimentation and is amenable for imaging. Through visualisation of calcium, Processing bodies and meiotic spindles, I show that osmotic pressure acts as an initiation cue for the calcium wave and downstream processes, including the resumption of cell cycle and the dispersion of the translational repression sites. I further show that aquaporin channels, together with external sodium ions, play a role in coordinating swelling of the oocyte in response to the osmotic pressure. I proceed to identify the requirement of internal calcium sources together with a dynamic actin cytoskeleton for a calcium wave to occur. Through co-visualisation of calcium and actin, I provide the first evidence that the calcium wave is followed by a wavefront of non-cortical F-actin at egg activation, which requires the calcium wave. Genetic analysis supports a model where changes in osmotic pressure trigger the calcium wave via stretch sensitive calcium channels in the oocyte membrane and the calcium wave is relayed by nearby channels via the actin cytoskeleton. My work concludes that the mechanism of egg activation in Drosophila is more similar to plants, compared to most vertebrates.

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.

High osmolarity causes amoebae of the cellular slime mould Polysphondylium pallidum to individually encyst, forming microcysts. During microcyst differentiation, actin is tyrosine phosphorylated. Tyrosine phosphorylation of actin is independent of encystment conditions and occurs during the final stages of microcyst formation. During microcyst germination, actin undergoes dephosphorylation prior to amoebal emergence. Renewed phosphorylation of actin in germinating microcysts can be triggered by increasing the osmolarity of the medium which inhibits emergence. Immunofluorescence reveals that actin is dispersed throughout the cytoplasm in dormant microcysts. Following the onset of germination, actin is observed around vesicles where it co-localizes with phosphotyrosine. Prior to emergence, actin localizes to patches near the cell surface. Increasing osmolarity disrupts this localization and causes actin to redistribute throughout the cytoplasm, a situation similar to that observed in dormant microcysts. Together, these results indicate an association between actin tyrosine phosphorylation, organization of the actin cytoskeleton, and microcyst dormancy.