Measurement of Outflow Facility Using iPerfusion

Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion^™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems.     

Generation of an Fsp1 (fibroblast‐specific protein 1)‐Flpo transgenic mouse strain

Recombination systems represent a major breakthrough in the field of genetic model engineering. The Flp recombinases (Flp, Flpe, and Flpo) bind and cleave DNA Frt sites. We created a transgenic mouse strain ([Fsp1‐Flpo]) expressing the Flpo recombinase in fibroblasts. This strain was obtained by random insertion inside mouse zygotes after pronuclear injection. Flpo expression was placed under the control of the promoter of Fsp1 (fibroblast‐specific protein 1) gene, whose expression starts after gastrulation at Day 8.5 in cells of mesenchymal origin. We verified the correct expression and function of the Flpo enzyme by several ex vivo and in vivo approaches. The [Fsp1‐Flpo] strain represents a genuine tool to further target the recombination of transgenes with Frt sites specifically in cells of mesenchymal origin or with a fibroblastic phenotype.     

Oxygen‐mediated plasticity confers hypoxia tolerance in a corallivorous polychaete

There is mounting evidence that the deoxygenation of coastal marine ecosystems has been underestimated, particularly in the tropics. These physical conditions appear to have far‐reaching consequences for marine communities and have been associated with mass mortalities. Yet little is known about hypoxia in tropical habitats or about the effects it has on reef‐associated benthic organisms. We explored patterns of dissolved oxygen (DO) throughout Almirante Bay, Panama and found a hypoxic gradient, with areas closest to the mainland having the largest diel variation in DO, as well as more frequent persistent hypoxia. We then designed a laboratory experiment replicating the most extreme in situ DO regime found on shallow patch reefs (3 m) to assess the response of the corallivorous fireworm, Hermodice carnaculata to hypoxia. Worms were exposed to hypoxic conditions (8 hr ~ 1 mg/L or 3.2 kPa) 16 times over an 8‐week period, and at 4 and 8 weeks, their oxygen consumption (respiration rates) was measured upon reoxygenation, along with regrowth of severed gills. Exposure to low DO resulted in worms regenerating significantly larger gills compared to worms under normoxia. This response to low DO was coupled with an ability to maintain elevated oxygen consumption/respiration rates after low DO exposure. In contrast, worms from the normoxic treatment had significantly depressed respiration rates after being exposed to low DO (week 8). This indicates that oxygen‐mediated plasticity in both gill morphology and physiology may confer tolerance to increasingly frequent and severe hypoxia in one important coral predator associated with reef decline.