Comparison of Different Cell Culture Media in the Model of the Isolated and Superfused Bovine Retina: Investigating the Limits of More Physiological Perfusion Solutions.

PURPOSE: The isolated superfused retina is a standardized tool in ophthalmological research. However, stable electroretinogram (ERG) responses can only be obtained for around eight hours; therefore, limiting its use. The aim of this study was to evaluate the short-term potential of different cell culture media and to promote long-term testing based on the results obtained. MATERIALS AND METHODS: For the experimental procedure bovine retinae were prepared and perfused with the standard Sickel solution and an ERG was performed. After recording stable a- or b-waves, different media (Dulbecco's Modified Eagle's Medium (DMEM), MACS, and Neurobasal) were superfused for 45 minutes. ERG recovery was monitored overall for 75 minutes. Analysis of the mRNA expression of Thy-1, GFAP, Bax/Bcl-2-ratio, Rhodopsin, and Opsin via qRT-PCR was performed directly after ERG recording on the same retina. RESULTS: None of the tested media had a negative effect on a-wave amplitudes, although b-wave amplitudes decreased (DMEM) or increased (MACS and Neurobasal) compared to the standard solution (Sickel) after 45 minutes of exposure. However, after 75 minutes of wash-out, no difference to the standard solution alone could be observed. Exposure to different media either had no effect or decreased the Opsin and Rhodopsin mRNA levels. Thy-1 expression was strongly diminished in DMEM and MACS (by 2-3-fold), whereas incubation in Neurobasal medium led to a slight increase compared to incubation with the standard solution. Furthermore, the Bax/Bcl-2 ratio indicated an anti-apoptotic effect (Bax/Bcl-2 = 0.16; p < 0.05) for Neurobasal. CONCLUSION: Neurobasal medium displayed the best electrophysiological properties in the short-term and may be applicable for stable long-term escalation testing.

Investigating short-term toxicity of melphalan in a model of an isolated and superfused bovine retina.

BACKGROUND: Melphalan, as a treatment for retinoblastoma, has been applied intra-arterially by catheterisation of the ophthalmic artery or intravitreally, aiming to reduce systemic side effects of intravenous drug therapy. This study evaluates retinal toxicity of different melphalan concentrations measured by electroretinogram (ERG) in an isolated and perfused retinal whole mount culture. METHODS: For functional testing, bovine retinas were prepared and perfused with an oxygen-saturated standard solution and the ERG was recorded until stable b-wave or a-wave amplitudes were reached. Thereafter, retinae were exposed to 80, 160 and 320 µg/ml of melphalan for 30 min. After exposure, a washout was performed thrice for 5 min each and the ERG amplitude recovery was monitored for 60 min. To investigate the effects on photoreceptor function, 1-mM asparate was added to suppress the b-wave and obtain isolated a-waves. RESULTS: While no toxic effects for a concentration of 80 µg/ml were observed, both b- and a-waves were significantly reduced after application of 160 (b-wave 43.8 %, p = 0.03; a-wave 28.2 %, p = 0.04) and 320 µg/ml (b-wave 20.0 %, p = 0.04; a-wave 35.8 %, p = 0.02). For 320 µg/ml, this reduction remained significant at the end of the washout (b-wave 40.0 % p = 0.02; a-wave 26.4 %, p = 0.02). CONCLUSIONS: Epiretinal or intraretinal concentrations of 80-µg/ml melphalan do not cause toxic effects in this in vitro model. Concentrations higher than 160 µg/ml should be avoided.

Melatonin inhibits insulin secretion in rat insulinoma ß-cells (INS-1) heterologously expressing the human melatonin receptor isoform MT2.

Melatonin exerts some of its effects via G-protein-coupled membrane receptors. Two membrane receptor isoforms, MT1 and MT2, have been described. The MT1 receptor is known to inhibit second messenger cyclic adenosine monophosphate (cAMP) signaling through receptor-coupling to inhibitory G-proteins (G(i) ). Much less is known about the MT2 receptor, but it has also been implicated in signaling via G(i) -proteins. In rat pancreatic ß-cells, it has recently been reported that the MT2 receptor plays an inhibitory role in the cyclic guanosine monophosphate (cGMP) pathway. This study addresses the signaling features of the constitutively expressed human recombinant MT2 receptor (hMT2) and its impact on insulin secretion, using a rat insulinoma ß-cell line (INS-1). On the basis of a specific radioimmunoassay, insulin secretion was found to be more strongly reduced in the clones expressing hMT2 than in INS-1 controls, when incubated with 1 or 100 nm melatonin. Similarly, cAMP and cGMP levels, measured by specific enzyme-linked immunosorbent assays (ELISAs), were reduced to a greater extent in hMT2 clones after melatonin treatment. In hMT2-expressing cells, the inhibitory effect of melatonin on insulin secretion was blocked by pretreatment with pertussis toxin, demonstrating the coupling of the hMT2 to G(i) -proteins. These results indicate that functional hMT2 expression leads to the inhibition of cyclic nucleotide signaling and a reduction in insulin release. Because genetic variants of the hMT2 receptor are considered to be risk factors in the development of type 2 diabetes, our results are potentially significant in explaining and preventing the pathogenesis of this disease.

Lupin protein compared to casein lowers the LDL cholesterol:HDL cholesterol-ratio of hypercholesterolemic adults.

BACKGROUND: Lupin protein had hypocholesterolemic effects in laboratory animals. However, the effect in humans has not been elucidated till now. AIM OF THE STUDY: To investigate the effect of lupin protein on circulating cholesterol in plasma and lipoproteins of hypercholesterolemic subjects. SUBJECTS AND METHODS: A randomised, double-blind, placebo-controlled, parallel trial (23 females and 20 males completed the trial) was conducted to compare the effects of lupin protein versus casein as control protein on plasma lipids and amino acids. Thirty-five grams of the test protein were consumed daily for 6 weeks. RESULTS: Both lupin protein and casein resulted in a reduction of circulating plasma cholesterol (-0.50 +/- 0.64 and -0.47 +/- 0.79 mM; P < 0.05) from baseline to week 6. The reduction of plasma cholesterol was mainly caused by a reduction of LDL cholesterol in the lupin protein group (-0.31 +/- 0.46 mM; P < 0.05), while in the casein group HDL cholesterol significantly declined (-0.17 +/- 0.15 mM; P < 0.05). Comparing the lupin protein group with the casein group yielded a difference in the net changes from baseline to week 6 in the LDL:HDL cholesterol-ratio of -0.24 (95% CI: -0.007, -0.479; P < 0.05). No significant differences in net changes were observed for plasma concentrations of triglycerides, glucose, homocysteine, taurine and most of the amino acids. CONCLUSIONS: Lupin protein compared to casein slightly lowered the concentration of LDL cholesterol in hypercholesterolemic subjects, without altering HDL cholesterol. No or minor effects of lupin protein were observed on circulating glucose, homocysteine and plasma amino acids.