Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.     

Effect of natural exogenous antioxidants on aging and on neurodegenerative diseases

Abstract

Aging and neurodegenerative diseases share oxidative stress cell damage and depletion of endogenous antioxidants as mechanisms of injury, phenomena that are occurring at different rates in each process. Nevertheless, as the central nervous system (CNS) consists largely of lipids and has a poor catalase activity, a low amount of superoxide dismutase and is rich in iron, its cellular components are damaged easily by overproduction of free radicals in any of these physiological or pathological conditions. Thus, antioxidants are needed to prevent the formation and to oppose the free radicals damage to DNA, lipids, proteins, and other biomolecules. Due to endogenous antioxidant defenses are inadequate to prevent damage completely, different efforts have been undertaken in order to increase the use of natural antioxidants and to develop antioxidants that might ameliorate neural injury by oxidative stress. In this context, natural antioxidants like flavonoids (quercetin, curcumin, luteolin and catechins), magnolol and honokiol are showing to be the efficient inhibitors of the oxidative process and seem to be a better therapeutic option than the traditional ones (vitamins C and E, and β-carotene) in various models of aging and injury in vitro and in vivo conditions. Thus, the goal of the present review is to discuss the molecular basis, mechanisms of action, functions, and targets of flavonoids, magnolol, honokiol and traditional antioxidants with the aim of obtaining better results when they are prescribed on aging and neurodegenerative diseases.     

Ascorbic acid prolongs the viability and stability of isolated perfused lungs: A mechanistic study using 31P and hyperpolarized 13C nuclear magnetic resonance

Ex vivo lung perfusion (EVLP) has recently shown promise as a means of more accurately gauging the health of lung grafts and improving graft performance post-transplant. However, reperfusion of ischemic lung promotes the depletion of high-energy compounds and a progressive loss of normal mitochondrial function, and it remains unclear how and to what extent the EVLP approach contributes to this metabolic decline. Although ascorbate has been used to mitigate the effects of ischemia–reperfusion injury, the nature of its effects during EVLP are also not clear. To address these uncertainties, this study monitored the energy status of lungs during EVLP and after the administration of ascorbate using ³¹P and hyperpolarized ¹³C NMR (nuclear magnetic resonance). Our experiments demonstrated that the oxidative phosphorylation capacity and pyruvate dehydrogenase flux of lungs decline during ex vivo perfusion. The addition of ascorbate to the perfusate prolonged lung viability by 80% and increased the hyperpolarized ¹³C bicarbonate signal by a factor of 2.7. The effect of ascorbate is apparently due not to its antioxidant quality but rather to its ability to energize cellular respiration given that it increased the lung’s energy charge significantly, whereas other antioxidants (glutathione and α-lipoic acid) did not alter energy metabolism. During ascorbate administration, inhibition of mitochondrial complex I with rotenone depressed energy charge and shifted the metabolic state of the lung toward glycolysis; reenergizing the electron transport chain with TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) recovered metabolic activity. This indicates that ascorbate slows the decline of the ex vivo perfused lung’s mitochondrial activity through an independent interaction with the electron transport chain complexes.     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

The use of clip cages to restrain insects reduces leaf expansion systemically in Rumex obtusifolius

Abstract. 1. Clip cages have been used widely by experimental ecologists to contain insects on plants.
2. Under controlled conditions, the effect of applying clip cages alone and clip cages and the chrysomelid beetle Gastrophysa viridula on systemic leaf expansion of Rumex obtusifolius was investigated. Treatments were applied to the fully expanded fourth leaf and expansion of leaf 8 was measured over a period of 22 days.
3. The application of clip cages reduced the rate at which leaf area increased and led to reductions in final leaf areas.
4. Clip cages have systemic effects on plant development and these effects are sustained even after the clip cage is removed. Investigators should take this into account in designing experiments.     

Frost Injury as a Possible Inciting Factor in Bud and Shoot Necroses of Fraxinus excelsior L.

Large numbers of European ash have died in Poland in all age classes during the last ten years. The characteristic symptom occurring on shoots of planted and self‐sown seedlings was bark necroses starting from the shoot apex, necrotic buds, or leaf and twig scars. The results showed that in the bud tissue of cold acclimated European ash extracellular and intracellular ice formation occurred at approximately ?9 and ?32°C, respectively. In deacclimated plants in spring water supercooling is limited by the heterogenous ice nucleation temperature and consequently the cold tolerance is ?9 to ?4°C for bud tissues and ?13 to ?9°C for shoots. Isolations of fungi were performed from dead buds and from necroses occurring on the main stem. Alternaria alternata, Fusarium lateritium and Phomopsis scobina were among the fungi occurring in both these organs at frequencies of more than 7%. Cylindrocarpon heteronemum, Diplodia mutila and Tubercularia vulgaris from necroses were only isolated in frequencies; 3.3, 1.2 and 5.4%, respectively. It seems likely that freezing injury is the inciting factor, which combined with fungal colonization manifests itself as fatal damage to European ash buds and shoots.     

Localization of vanadium-containing particles in the lungs of uranium/vanadium miners

Several geological formations of the Utah-Colorado mining region mined for uranium ore during and after World War II had been mined earlier for vanadium. Therefore, most miners and millers from that region were exposed to those metals’ ores or tailings at one time or another. Preliminary investigation to determine uranium and vanadium retained in the lungs of a former uranium miner and miller from this region, who died of lung cancer (mesothelioma), showed a high nonuniform distribution of vanadium. This observation led to the hypothesis that the vanadium content in the lungs could be associated with inhaled particles. Further examination of spectra of characteristic X-rays obtained by scanning particle-induced X-ray emission (microPIXE) of an autopsy sample of this lung indicated that vanadium was indeed present in localized sites within the 20-μm spatial resolution of the proton beam. This work points out that the microPIXE-RBS (Rutherford backscattering) test for vanadium can be used for site localization of inhaled particles retained in the lungs. Further studies are in progress to: (i) locate uranium-bearing particles in lung tissues of former uranium miners and millers; and (ii) evaluate the local doses of alpha radiation received from these particles.     

Insect-induced chemical changes in young lodgepole pine (Pinus contorta): the effect of previous defoliation on oviposition, growth and survival of the pine beauty moth, Panolis flammea

ABSTRACT. 1. Significantly more eggs were laid by adult female pine beauty moths ( Panolis flammea (D&S)) on previously undefoliated lodgepole pine ( Pinus contorta Douglas) saplings than on those which had been partially defoliated the year before.
2. The larval weights, mean relative growth rates and survival rates of P.flammea were lower on defoliated trees than on undefoliated trees.
3. Marked and significant differences between the monoterpene profiles of the shoot cortical oleoresin of defoliated and undefoliated trees were linked to oviposition preferences in P.flammea.
4. Differences occurred between the levels of soluble tannins in previously defoliated and undefoliated trees.
5. The results are discussed in relation to earlier work on insect-induced changes in plants, and the significance of inter-provenance differences in lodgepole pine response is assessed.