Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Importance of habitat patch size for occupancy and density of aspen-associated saproxylic beetles

In Fennoscandian boreal forests, aspen (Populus tremula) is one of the most important tree species for biodiversity. In this study we explore how occupancy and density of beetles associated with dead aspen are related to habitat patch size and connectedness in a 45,000 ha boreal managed forest landscape in central Sweden. Patch size was estimated as amount of breeding substrate and connectedness as crown cover of living aspen in the surrounding landscape. The beetles were sampled by sieving of bark or by inspection of species-characteristic galleries in 56 patches with dead aspen. Six of nine aspen-associated species (Xylotrechus rusticus, Ptilinus fuscus, Mycetophagus fulvicollis, Cyphaea curtula, Homalota plana and Endomychus coccineus) showed a positive significant relationship between habitat patch size and occupancy. For all these species, except C. curtula, there was also a significant positive relationship between patch size and density. Connectedness was not retained as a significant variable in the analyses. Species not defined as aspen-associated constituted a significantly larger proportion of the total density of individuals of saproxylic beetles in smaller habitat patches than in larger patches. Richness of aspen-associated species was positively related to habitat patch size. Efforts in the managed forest should be directed towards preserving and creating larger patches of living and dead aspen trees and increasing the amount of aspen at the landscape level.     

Islands as refugia of <Emphasis Type="Italic">Trifolium repens</Emphasis> genetic diversity

Island populations are often thought to be more susceptible to the loss of genetic diversity as a consequence of limited population size and genetic drift, greater susceptibility to detrimental stochastic events and low levels of immigration. However the geographic isolation of islands may create refuges for native crop species whose genetic diversity is threatened from the genetic erosion occurring in mainland areas as a result of crop-wild gene flow and genetic swamping. Many UK islands remain uncharacterised in terms of plant genetic diversity. In this study we compared the genetic diversity of mainland populations and landraces of Trifolium repens with wild populations collected from the islands surrounding the UK, including the island of Hirta in the St Kildan archipelago. Individuals from St Kilda represent a unique conservation resource, with populations both highly differentiated from UK mainland populations and genetically distinct from cultivated varieties, whilst able to retain diversity through limited human influence on the islands. In contrast, there is relative genetic similarity of wild UK populations to cultivated forms highlighted in mainland populations, but with geographic barriers preventing complete homogenisation of the mainland UK genepool. We underline the need for conservation priorities to include common species that are threatened by gene flow from cultivation, and draw attention to the potential of islands to preserve natural levels of genetic diversity.     

Alpine flora dynamics – a critical review of responses to climate change in the Swedish Scandes since the early 1950s

Reports about changes of alpine plant species richness over the past 60 years in the Swedish Scandes are reviewed, synthesized and updated with data from recent reinventories. Methodologically, this endeavour is based on resurveys of the floristic composition on the uppermost 20 m of four high‐mountain summits. The key finding is that the species pool has increased by 60–170% since the 1950s and later. Some of the invading species are new to the alpine tundra, with more silvine and thermophilic properties than the extant alpine flora. Not a single species of the original flora has disappeared from any of the summits. This circumstance is discussed in perspective of widespread expectations of pending temperature‐driven extinction of alpine species in an alleged future warmer climate. These progressive changes coincided with distinct warming (summer and winter) since the late 1980s. During a short cooler period (1974–1994), the species numbers decreased and the upper elevational limits of some ground cover species descended. Thus, discernible responses, concurrent with both warming and cooling intervals, sustain a strong causal link between climate variability and alpine plant species richness. Methodologically, plot‐less revisitation studies of the present kind are beset with substantial uncertainties, which may overstate floristic changes over time. However, it is argued here that carefully executed and critically interpreted, no other method can equally effectively sense the earliest phases of plant invasions into alpine vegetation.     

Activation of VPAC1 receptors aggravates early atherosclerosis in hypercholesterolemic apolipoprotein E-deficient mice

Objective: Vasoactive intestinal peptide (VIP) is a 28-amino acid peptide widely expressed in the body and binding three types of receptors: VPAC₁-R, VPAC₂-R and PAC₁-R. Based on beneficial effects of VIP and VPAC₁-R agonists in mouse models of several chronic inflammatory disorders, we hypothesized that activation of VIP receptors would prevent atherosclerosis development in apolipoprotein E-deficient mice.Methods and results: Contrary to our hypothesis, administration of a VPAC₁-R agonist, (Ala^11,22,28)-VIP aggravated atherosclerotic lesion development in the aortic root of these mice compared to control mice. This was accompanied by a significant increase in the expression of MHC class II protein I-A^b, and suggests enhanced inflammatory activity in the vessel wall. The amount of macrophage-specific CD68 staining as well as serum cholesterol and triglyceride levels did not change as a result of the (Ala^11,22,28)-VIP treatment, i.e. the treatment resulted in significant changes in lipid accumulation in the lesions without changing the number of macrophages or systemic lipid levels. Interestingly, administration of VIP did not alter the course of the disease.Conclusion: Despite beneficial effects in murine models of several inflammatory disorders, VPAC₁-R activation aggravates atherosclerotic lesion formation in apolipoprotein E-deficient mice through enhanced inflammatory activity in the vessel wall.     

Characterization of oil and starch accumulation in tubers of Cyperus esculentus var. sativus (Cyperaceae): A novel model system to study oil reserves in nonseed tissues

? Premise of the study: Storage oil (triacylglycerol) accumulates in tissues such as the embryo and endosperm of seeds and the fruit mesocarp, but seldom in underground organs. As a rare exception, cultivated variants of yellow nutsedge (Cyperus esculentus) contain high amounts of both oil and starch in the mature tubers. ? Methods: Biochemical analyses and light and electron microscopy were used to study the accumulation patterns of storage nutrients in developing nutsedge tubers. ? Key results: During the initial phase of tuber development, the conducting rhizome tissue is transformed into a storage compartment, then massive storage reserves accumulate in the tuber. At the beginning of tuber development, a large sugar load coincided with the onset of starch accumulation. Oil accumulation started later, concomitant with a substantial drop in the sugar content. Initially, oil accumulated at a lower rate compared to starch, but the rate later increased; after 6 wk, oil made up 24% of tuber dry mass, while starch made up 32%. Protein concentration changed only a small amount throughout this development. Oil and starch accumulated in the same cells throughout the tubers in a sequential fashion during tuber development. ? Conclusions: The developmental pattern in the build up of storage nutrients in the tubers highlights nutsedge as a novel model plant, having potential to significantly widen our understanding on how synthesis of storage reserves, and in particular oils, is regulated and directed in nonseed tissues such as tubers and roots.     

Immigration of Picea abies into North-Central Sweden. New evidence of regional expansion and tree-limit evolution

Analyses of subfossil tree remains in peats and raw humus soils account for the immigration and spread of Picea abies (Norway spruce) into Sweden and the evolution of the alpine tree-limit ecotone. Picea abies is recorded for the first time about 11 000 BP, on an early emerging nunatak in the southern Swedish Scandes. Prior to c. 8 000 BP, Picea was strictly bound to high elevations in the west. Farther to the east in North-Central Sweden, Picea emerged in the subfossil record mainly after c. 6 000 BP. Later on, growth of local founder populations and landscape-scale expansion may have been forced chiefly by a successively less seasonal climate (the Milankovitch model of orbital forcing), promoting increased net soil moisture and possibly a deeper and more persistent snow cover. Already at the Weichselian/Holocene transition, an elevational tree-limit ecotone was established and arborescent Picea grew at least 400 m higher than the modern tree-limit. Until about 8 000 BP, the species-limit descended, whereafter it stabilized up to the present day. Lack of significant species-limit retraction after c. 8 000 BP may seem paradoxical in perspective of independently inferred climate cooling in response to reduced insolation and land uplift. This could be a consequence of substantial phenotypic plasticity and increased snow accumulation, mitigating the long-term cooling. Thus, the elevational species-limit (krummholz) of Picea abies is out of equilibrium with the modern thermal climate. The striking incongruence between the results exposed here and earlier palynological interpretations of the same biogeographical process will have implications for the use of pollen data for range-limit reconstructions within historical biogeography, and urges for re-evaluation of certain aspects of the Fennoscandian forest history.