Limits of the confocal laser-scanning technique in measurements of time-resolved autofluorescence of the ocular fundus]

GOAL: The 2-dimensional measurement of the time-dependent autofluorescence has in combination of confocal laser scanner technique and time correlated single photon counting the potential for the investigation of the metabolism state at the eye ground. It was to be examined, to what extent these measurements are influenced through the auto-fluorescence of the crystalline lens. MATERIAL AND METHOD: The time-dependent auto-fluorescence was measured on eyes of 21 patients with age-related macular degeneration (AMD) and of 26 healthy subjects in 40 degrees fundus images. The experimental set-up used, was developed at the eye clinic of the University of Jena. In this scanning laser ophthalmoscope, the fundus was excited at 446 nm by pulses of 100 ps full width at half maximum and 40 MHz repetition rate. The auto-fluorescence was detected with a time resolution of 25 ps for wavelengths > 500 nm. The dynamic auto-fluorescence of each pixel was approximated by a tri-exponential model function. In order to determine the influence of the fluorescence of the crystalline lens, healthy eyes and eyes were compared with implanted artificial intra-ocular lenses. For comparison the frequency distributions were used, which the decay times t1, t2 and t3 in the fundus images had been calculated with. RESULTS: Clear differences were shown in the frequency distributions of the fluorescence lifetimes between AMD-patients and healthy subjects, It resulted, however, from a crucial analysis that the fluorescence of the crystalline lens is not suppressed by the confocal laser scanning principle sufficiently. In particular the long lifetime t3 is overlapped by the fluorescence of the crystalline lens of about 4.6 ns. To the complete suppression of the influence of the lens fluorescence in measurements of the time-resolved fundus auto-fluorescence it is suggested to combine the confocal laser scanning technique with the principle of the aperture diaphragm division. CONCLUSION: In order to be able to determine the capability of measurements of the time-dependent fundus auto-fluorescence as diagnostic tool exactly, the influence of the crystalline lens is to reduce through the combination of confocal laser scanning ophthalmoscopy with the principle of the aperture diaphragm division diversely.     

Glycation of fibrinogen in diabetic patients: a practical colorimetric assay

We have developed a practical method for the measurement of glycated fibrinogen which combines purification by glycine precipitation with a nitroblue tetrazolium assay at 56°C in the presence of Zwittergent 3–14. This detergent, at a concentration of 10 g/l, was chosen because it combines solubilization of fibrinogen and low coloration of blanks while increasing the sensitivity of the assay. A positive correlation (r=0.85,P<0.02) was found between this procedure and a previously reported one based on the thiobarbituric acid assay. To validate the method we then measured fibrinogen, glycated fibrinogen, serum fructosamine and erythrocyte glycated haemoglobin in a population of healthy euglycaemic subjects (n=30) and a population of diabetic patients (n=40). Glycated fibrinogen was significantly higher in diabetic patients than in control subjects (15.42±0.70vs 11.52±0.27 nmol of deoxymorpholinofructose equiv. per mg,P<0.005). Given the short half-life of fibrinogen, assay of its glycation may be useful as a short-term marker of glycaemic control.     

The influence of various housing conditions on the ocular fundus in Wistar rats

Examinations of ocular fundi were performed on male Wistar rats which were housed for 2 years individually or in groups of 3, 5 or 10 animals per cage. Photographs were taken to make the data objective and provide a permanent record. The results indicated no differences between the various groups, although there were marked differences between individual rats.     

An association between fibrinogen gene polymorphisms and diabetic peripheral neuropathy in young patients with type 1 diabetes

Molecular Biology Reports - In complex etiopathogenesis of diabetic peripheral neuropathy (DPN), hemostatic dysfunction and subclinical inflammation play a possible role. Fibrinogen is involved in...     

Evaluation of oxidative stress markers and vascular risk factors in patients with diabetic peripheral neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most common diabetic chronic complications. The pathogenesis of DPN is complex and involves an intertwined array of mechanisms. The purposes of this study were to evaluate the association of oxidative stress and vascular risk factors with the prevalence of DPN and to determine the role of these biochemical parameters in the prognosis of DPN. One hundred patients with type 2 diabetes mellitus and 40 clinically healthy individuals were evaluated. The patients were divided into two groups. Group 1 included 40 diabetic patients without peripheral neuropathy, and group 2 consisted of 60 patients with DPN. Erythrocytes glutathione (GSH) level, plasma malondialdehyde (MDA), nitrite/nitrate (NOx) and homocysteine (Hcy) levels as well as serum ceruloplasmin (Cp), total antioxidants (TAO), endothelin-1 (ET-1) levels and γ-glutamyl transferase (GGT) activity were estimated. A significant decrease of erythrocyte GSH was observed in groups 1 and 2 relative to the controls. An increase in glycosylated haemoglobin (HbA1c), MDA, NOx, GGT, Cp, TAO, Hcy and ET-1 was noted in patients with DPN. In conclusion, oxidative stress biomarkers and vascular risk factors could be important in the pathogenesis of DPN. The measurement of serum GGT and Hcy in addition to HbA1c and disease duration could facilitate the early detection of neuropathy in diabetic patients.     

Analysis of fibrinogen genes in patients with congenital afibrinogenemia

Several cDNA clones coding for A alpha, B beta and gamma chains of fibrinogen have been isolated from a human liver cDNA library. They were selected by differential hybridization with probes raised against fractionated liver mRNA (positive probes) and muscle and albumin mRNA (negative probes), then firmly identified by positive hybridization selection. Three of these clones, encoding A alpha, B beta and gamma fibrinogen chain sequences, were further characterized by restriction mapping and used as probes to characterize fibrinogen mRNAs from adult and fetal liver and fibrinogen genes in normal individuals and two afibrinogenemic patients. The results indicate that there is a single copy of the fibrinogen genes which are present and grossly intact in afibrinogenemic DNA.     

Assessment of non-mydriatic fundus photography in detection of diabetic retinopathy.

Non-mydriatic retinal photography with later interpretation of the photographs was assessed as a screening method for the detection of diabetic retinopathy; when compared with an ophthalmologist''s clinical assessment in a random group of 62 diabetic patients it was accurate (false negative 6.8%, false positive 2%) and sensitive (sensitivity 96%, specificity 98%). The assessment of further management required based on analysis of the photographs was 96.5% in agreement with the further management suggested by the ophthalmologist after direct clinical assessment of the patient. If this technique were used to screen patients in a typical diabetic clinic the predicted positive accuracy rate would be 84% and the predicted negative accuracy rate 99.5%.     

Normal color variations of the canine ocular fundus,a retrospective study in Swedish dogs

Background

Anthelmintic treatment is the most common way of controlling nematode infections in ruminants. However, several countries have reported anthelmintic resistance (AR), representing a limitation for sustainable small ruminant production. The knowledge regarding worm control management represents a baseline to develop a guideline for preventing AR. The aim of the present study was therefore to improve our knowledge about the worm control practices in small ruminant flocks in Norway.

Methods

A questionnaire survey regarding worm control practices was performed in small ruminant flocks in Norway. Flocks were selected from the three main areas of small ruminant farming, i.e. the coastal, inland and northern areas. A total of 825 questionnaires, comprising 587 sheep flocks (return rate of 51.3%) and 238 goat flocks (52.6%) were included.

Results

The results indicated that visual appraisal of individual weight was the most common means of estimating the anthelmintic dose used in sheep (78.6%) and goat (85.1%) flocks. The mean yearly drenching rate in lambs and ewes were 2.5 ± 1.7 and 1.9 ± 1.1, respectively, whereas it was 1.0 (once a year) in goats. However, these figures were higher in sheep in the coastal area with a rate of 3.4 and 2.2 in lambs and ewes, respectively. Benzimidazoles were the predominant anthelmintic class used in sheep flocks (64.9% in 2007), whereas benzimidazoles and macrocyclic lactones were both equally used in dairy goat flocks. In the period of 2005-2007, 46.3% of the sheep flocks never changed the anthelmintic class. The dose and move strategy was practiced in 33.2% of the sheep flocks.

Conclusions

The present study showed that inaccurate weight calculation gives a risk of under-dosing in over 90% of the sheep and goat flocks in Norway. Taken together with a high treatment frequency in lambs, a lack of anthelmintic class rotation and the common use of a dose-and-move strategy, a real danger for development of anthelmintic resistance (AR) seems to exist in Norwegian sheep and goat flocks. This risk seems particularly high in coastal areas where high treatment frequencies in lambs were recorded.     

Blood-soluble Fas levels are increased in type 2 diabetic patients with peripheral vascular disease.

AIM: To investigate the possible utility of plasma sFas (soluble Fas) levels as a marker of peripheral vascular disease (PVD) in type 2 diabetic patients, and the relationship between classical cardiovascular risk factors and sFas levels in these patients. MATERIALS AND METHODS: sFas levels were measured in 57 type 2 diabetic patients with and 60 without PVD matched for age and sex. Diagnosis of PVD was established in presence of at least one of the following criteria: leg or foot amputation of vascular cause, lower-extremity arterial angioplasty or surgical by-pass, or ankle-braquial index (ABI) less than 1 in at least one side of the body. ELISA was used to measure sFas levels. RESULTS: None of the risk factors assessed total cholesterol, HDL cholesterol, triglycerides, CRP, ACE, fibrinogen, Lp(a) and homocysteine was significantly different between both groups of patients. However, patients with PVD had higher plasma sFas levels than the group without PVD (10.25+/-3.7 ng/ml VS. 8.86+/-2.6 ng/ml; p=0.02). Levels of sFas were 1.45 ng/ml (95% CI: 0.32-2.58; p=0.013) higher in PVD patients when adjusting by age, total, HDL and LDL cholesterol, triglycerides, homocysteine, CRP, ACE, arterial hypertension and tobacco smoking. Using multiple logistic regression sFas is a predictor of PVD, although not potent. CONCLUSION: Plasma sFas may be an independent marker of PVD in type 2 diabetes mellitus patients.     

A comparison of the fibrinogen receptor distribution on adherent platelets using both soluble fibrinogen and fibrinogen immobilized on gold beads

The distribution of fibrinogen receptors was determined on the surface of adherent platelets using both direct labeling with the ligand fibrinogen which was immobilized on gold particles (Fg-Au) and indirect immunogold (Ig-Au) labeling of bound soluble fibrinogen identified with a rabbit polyclonal anti-fibrinogen antibody. Two distinctly different patterns of labeling were obtained and appeared to depend on whether solid phase fibrinogen (Fg-Au) or soluble phase released fibrinogen were bound to the membrane receptor. The membrane-bound Fg-Au reorganized in patterns that closely mimicked the organization of the underlying cytoskeleton. In approximately 18% of the adherent platelets, Fg-Au was seen in channels or vesicle-like structures lying deep to the platelet surface suggesting internalization into the open canalicular system and/or endocytosis. The labeling pattern obtained when identifying the location of membrane-bound soluble released fibrinogen by Ig-Au was diffuse and lacked the organizational patterns characteristic of Fg-Au. Unlike the Fg-Au probe, early dendritic platelets were heavily labeled by the soluble phase fibrinogen using the Ig-Au technique. Although the label covered the entire exposed platelet membrane in fully spread platelets, labeling over the peripheral web was more dense than that over the intermediate or granulomere zone. The diffuse organization and heavier peripheral distributional pattern of the glycoprotein IIb-IIIa (GP IIb-IIIa) receptor in fixed, adherent platelets, was also seen with the GP IIb-IIIa receptor-specific antibody AP-2. The binding of both the Fg-Au and Ig-Au were inhibited using the tetrapeptide Arg-Gly-Asp-Ser (RGDS) (93% and 98% inhibition, respectively), AP-2 (98% and 97%, respectively) and platelets from patients with Glanzmann's thrombasthenia (GT) (99% and 98%, respectively). The data presented provides the first report that receptor reorganization, following binding of fibrinogen, appears to be related to the state of the ligand. Substrate bound fibrinogen (i.e., Fg-Au or fibrinogen bound to another platelet) induces receptor translocation toward the platelet granulomere in a capping-like phenomenon. On the other hand, the binding of soluble released fibrinogen results in formation of microclusters and short linear arrays in a diffuse distribution but does not induce central movement of receptors. Furthermore, double labeling studies clarify that Fg-Au does not identify all available fibrinogen receptors as many are occupied by soluble released fibrinogen. The data presented provides an interesting new perspective on what constitutes an appropriate ligand-receptor stimulus sufficient to induce receptor reorganization.