Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors.

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Dynamics of extracellular polymeric substances in UASB and EGSB reactors treating medium and low concentrated wastewaters.

In this work the dynamic study of EPS (Extracellular Polymeric Substances) concentration and distribution during the operation of two different reactor configurations (UASB and EGSB) is presented, treating medium (5 g COD/l) and low-concentrated (0.5 g COD/l) wastewater. Medium-concentrated wastewater was supplied for granules maturation as well as for stabilisation of the process. The effect of substrate change on granule characteristics was followed in both reactors. Total concentration of EPS associated to steady operation, was higher in the UASB reactor. The change to a low-concentrated substrate led to an increased difference, promoting a sharp destabilisation of the EGSB reactor, observing an increment in filamentous structures, causing biomass flotation and wash out. Although total concentration of EPS remained almost constant in the UASB reactor, their composition and distribution presented significant differences. The ratio of protein/polysaccharides as well as acidic-polysaccharides/total (neutral + acidic) polysaccharides decreased drastically in the EGSB reactor, while in the UASB reactor, the decrease was not so important and not enough for destabilisation of granule structure. Moreover, polysaccharides distribution seemed to have an important role in granule stability being enough to maintain granule cohesion only in the case of the UASB reactor. These observations point to composition and distribution of EPS rather than their total concentration as key parameters for granule stability and settleability.