Preconditioning with ethanol prevents postischemic leukocyte-endothelial cell adhesive interactions

Long-term ethanol consumption at low to moderate levels exerts cardioprotective effects in the setting of ischemia and reperfusion (I/R). The aims of this study were to determine whether 1) a single orally administered dose of ethanol [ethanol preconditioning (EtOH-PC)] would induce a biphasic temporal pattern of protection (early and late phases) against the inflammatory responses to I/R and 2) adenosine and nitric oxide (NO) act as initiators of the late phase of protection. Ethanol was administered as a bolus to C57BL/6 mice at a dose that achieved a peak plasma concentration of ~45 mg/dl 30 min after gavage and returned to control levels within 60 min of alcohol ingestion. The superior mesenteric artery was occluded for 45 min followed by 60 min of reperfusion beginning 10 min or 1, 2, 3, 4, or 24 h after ethanol ingestion, and the numbers of fluorescently labeled rolling and firmly adherent (stationary) leukocytes in single postcapillary venules of the small intestine were quantified using intravital microscopic approaches. I/R induced marked increases in leukocyte rolling and adhesion, effects that were attenuated by EtOH-PC 2-3 h before I/R (early phase), absent when assessed after 10 min, 1 h, and 4 h of ethanol ingestion, with an even more powerful late phase of protection reemerging when I/R was induced 24 h later. The anti-inflammatory effects of late EtOH-PC were abolished by treatment with adenosine deaminase, an adenosine A(2) (but not A(1)) receptor antagonist, or a NO synthase (NOS) inhibitor during the period of EtOH-PC. Preconditioning with an adenosine A(2) (but not an A(1)) receptor agonist in lieu of ethanol 24 h before I/R mimicked the protective actions of late phase EtOH-PC. Like EtOH-PC, the effect of preconditioning with an adenosine A(2) receptor agonist was abrogated by coincident NOS inhibition. These findings suggest that EtOH-PC induces a biphasic temporal pattern of protection against the proinflammatory effects of I/R. In addition, our observations are consistent with the hypothesis that the late phase of EtOH-PC is triggered by NO formed secondary to adenosine A(2) receptor-dependent activation of NOS during the period of ethanol exposure.     

A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo

Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.     

Diet of bluegill Lepomis macrochirus in a South African reservoir during winter and summer

Alien fishes are considered a major threat to aquatic biodiversity in South Africa, yet relatively little regional information on their biology and ecology is available for many of these species. Seasonal changes in the diet of the bluegill Lepomis macrochirus in Howieson’s Poort Dam, Grahamstown, were assessed during summer and winter in 2014–2015, using stomach content analysis. In winter, juvenile and adult fish diets were dominated by crustacean zooplankton and insects, respectively. In summer, juvenile fish fed on crustaceans and insects, whereas adults consumed mostly fish eggs, indicating a potential impact by these invasive fish on native fish through oophagy.     

Distribution of MT1 Melatonin Receptor Promoter-Driven RFP Expression in the Brains of BAC C3H/HeN Transgenic Mice

The pineal hormone melatonin activates two G-protein coupled receptors (MT₁ and MT₂) to regulate in part biological functions. The MT₁ and MT₂ melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT₁ melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT₁ promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT₁). RFP-MT₁ expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT₁ transgenic model provides a unique tool for studying the distribution of the MT₁ receptor in the brain of mice, its cell-specific expression and its function in vivo.     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.     

Clinical features of symptomatic patellofemoral joint osteoarthritis

Introduction

Patellofemoral joint osteoarthritis (OA) is common and leads to pain and disability. However, current classification criteria do not distinguish between patellofemoral and tibiofemoral joint OA. The objective of this study was to provide empirical evidence of the clinical features of patellofemoral joint OA (PFJOA) and to explore the potential for making a confident clinical diagnosis in the community setting.

Methods

This was a population-based cross-sectional study of 745 adults aged ≥50 years with knee pain. Information on risk factors and clinical signs and symptoms was gathered by a self-complete questionnaire, and standardised clinical interview and examination. Three radiographic views of the knee were obtained (weight-bearing semi-flexed posteroanterior, supine skyline and lateral) and individuals were classified into four subsets (no radiographic OA, isolated PFJOA, isolated tibiofemoral joint OA, combined patellofemoral/tibiofemoral joint OA) according to two different cut-offs: ''any OA'' and ''moderate to severe OA''. A series of binary logistic and multinomial regression functions were performed to compare the clinical features of each subset and their ability in combination to discriminate PFJOA from other subsets.

Results

Distinctive clinical features of moderate to severe isolated PFJOA included a history of dramatic swelling, valgus deformity, markedly reduced quadriceps strength, and pain on patellofemoral joint compression. Mild isolated PFJOA was barely distinguished from no radiographic OA (AUC 0.71, 95% CI 0.66, 0.76) with only difficulty descending stairs and coarse crepitus marginally informative over age, sex and body mass index. Other cardinal signs of knee OA - the presence of effusion, bony enlargement, reduced flexion range of movement, mediolateral instability and varus deformity - were indicators of tibiofemoral joint OA.

Conclusions

Early isolated PFJOA is clinically manifest in symptoms and self-reported functional limitation but has fewer clear clinical signs. More advanced disease is indicated by a small number of simple-to-assess signs and the relative absence of classic signs of knee OA, which are predominantly manifestations of tibiofemoral joint OA. Confident diagnosis of even more advanced PFJOA may be limited in the community setting.     

Isoform-selective 5'-AMP-activated protein kinase-dependent preconditioning mechanisms to prevent postischemic leukocyte-endothelial cell adhesive interactions

We previously demonstrated that preconditioning induced by ethanol consumption at low levels [ethanol preconditioning (EPC)] or with 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR-PC) 24 h before ischemia-reperfusion prevents postischemic leukocyte-endothelial cell adhesive interactions (LEI) by a mechanism that is initiated by nitric oxide formed by endothelial nitric oxide synthase. Recent work indicates that 1) ethanol increases the activity of AMP-activated protein kinase (AMPK) and 2) AMPK phosphorylates endothelial nitric oxide synthase at the same activation site seen following EPC (Ser1177). In light of these observations, we postulated that the heterotrimeric serine/threonine kinase, AMPK, may play a role in triggering the development of the anti-inflammatory phenotype induced by EPC. Ethanol was administered to C57BL/6J mice by gavage in the presence or absence of AMPK inhibition. Twenty-four hours later, the numbers of rolling and adherent leukocytes in postcapillary venules of the small intestine were recorded using an intravital microscopic approach. Following 45 min of ischemia, LEI were recorded after 30 and 60 min of reperfusion or at equivalent time points in control animals. Ischemia-reperfusion induced a marked increase in LEI relative to sham-operated control mice. The increase in LEI was prevented by EPC, an effect that was lost with AMPK inhibition during the period of ethanol exposure. Studies conducted in AMPK α(1)- and α(2)-knockout mice suggest that the anti-inflammatory effects of AICAR are not dependent on which isoform of the catalytic α-subunit is present because a deficiency of either isoform results in a loss of protection. In sharp contrast, EPC appears to be triggered by an AMPK α(2)-isoform-dependent mechanism.     

Activated neutrophils increase microvascular permeability in skeletal muscle: role of xanthine oxidase

To determine the role of xanthine oxidase in the microvascular dysfunction produced by activated granulocytes, we examined the effect of xanthine oxidase depletion or inhibition on the increase in microvascular permeability produced by infusion of the neutrophil activator phorbol myristate acetate (PMA). Changes in vascular permeability were assessed by measurement of the solvent drag reflection coefficient for total plasma proteins (sigma) in rat hindquarters subjected to PMA infusion in xanthine oxidase-replete and -depleted animals, in animals pretreated with the xanthine oxidase inhibitor oxypurinol, and in animals depleted of circulating neutrophils by pretreatment with antineutrophil serum (ANS). Xanthine oxidase depletion was accomplished by administration of a tungsten-supplemented (0.7 g/kg diet) molybdenum-deficient diet. In animals fed the tungsten diet, muscle total xanthine dehydrogenase plus xanthine oxidase activity was decreased to less than 10% of control values. Estimates of sigma averaged 0.84 +/- 0.04 in control hindquarters, whereas PMA infusion was associated with a marked increase in microvascular permeability (decrease in sigma to 0.68 +/- 0.03). PMA infusion also caused an increase in the amount of the radical-producing oxidase form of xanthine oxidase (from 3.9 +/- 0.05 to 5.6 +/- 0.4 mU/g wet wt). ANS pretreatment attenuated this permeability increase (sigma = 0.77 +/- 0.04) and diminished the rise in xanthine oxidase activity (4.9 +/- 0.5 mU/g wet wt). Xanthine oxidase depletion with the tungsten diet or pretreatment with oxypurinol had no effect on this neutrophil-mediated microvascular injury (sigma = 0.69 +/- 0.06 and 0.67 +/- 0.03, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

An improved method using densitometry for evaluating severity of laser photocoagulation induced CNV

Laser photocoagulation induced choroidal neovascularization currently is the most effective model available for the study of this disease in terms of efficacy of new drugs and therapies. Previously, evaluating the extent of choroidal neovascularization using this model was time- consuming and required the use of experienced personnel. We describe a new method for simple and rapid evaluation of laser induced choroidal neovascularization using densitometry. Fluorescein angiograms of a laser photocoagulated rat eye were scanned into a computer. Densitometry software subsequently was used to calculate the severity of the laser lesions. The densitometry method proved effective for calculating the extent of laser induced choroidal neovascularization. In addition, this method was more rapid than visual evaluations and less likely to produce errors.