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Background: Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells., Methods: Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13., Results: An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan., Conclusions: We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.

The development of alternative therapies for melanoma treatment is of great interest as long-term tumour regression is not achieved with new targeted chemotherapies on selected patients. We previously demonstrated that radioiodinated heteroarylcarboxamide ([131I]ICF01012) induced a strong anti-tumoural effect by inhibiting both primary tumour growth and dissemination process in a B16BL6 melanoma model. In our study, we show that a single injection of [131I]ICF01012 (ranging from 14.8 to 22.2 MBq) was effective and associated with low and transient haematological toxicity. Concerning pigmented organs, cutaneous melanocytes and skin were undamaged. In 30% of treated animals, no histological alteration of retina was observed, and in the remaining 70%, damages were restricted to the optic nerve area. Using the Medical Internal Radiation Dose methodology, we determined that the absorbed dose in major organs is very low (<4 Gy) and that a delivery of 30 Gy to the tumour is sufficient for an effective anti-tumoural response. Molecular analyses of treated tumours showed a strong radiobiological effect with a decrease in proliferation, survival and pro-angiogenic-related markers and an increase in tumour suppressor gene expression, melanogenesis and anti-angiogenic markers. All these features are in accordance with a tumour cell death mechanism that mainly occurs by mitotic catastrophe and provide a better understanding of in vivo anti-tumoural effects of [131I] radionuclide. Our findings raise [131I]ICF01012 a good candidate for disseminated melanoma treatment and strongly support transfer of [131I]ICF01012 to clinical trial., (Copyright © 2013 UICC.)

Aim: To evaluate the in vitro effects of bevacizumab (Avastin®) on the electroretinogram (ERG) in rats using a model of isolated perfused retina ERG recording., Methods: Retinas were isolated from rat eyes and placed in a chamber continuously perfused with a nutrient solution. The ERG was recorded every 3 min. Once the ERG b-wave amplitude was at a steady state, bevacizumab was added at concentrations of 0.25 and 0.5 mg/ml to the perfusion medium for 3 h., Results: We observed no effect on ERG amplitudes or kinetics when bevacizumab was added to the perfusion medium. In addition, we found no significant differences in the survival curves of the b-wave and PIII wave during the application of bevacizumab between bevacizumab-exposed retinas and control retinas., Conclusions: We demonstrate that bevacizumab has no in vitro toxic effects on the ERG of isolated perfused rat retina. Our study supports the retinal safety of bevacizumab with respect to retinal function., (Copyright © 2011 S. Karger AG, Basel.)

We investigated the capacity of Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells to take up all-trans-retinol (ROL) (vitamin A) and to metabolize it into retinyl esters (RE). Cultures of RPE cells were established from RCS and control newborn rats. All-trans-ROL was delivered to the apical surface of the RPE monolayer. Retinoids were analyzed by high-performance liquid chromatography. The cellular retinol-binding protein type I (CRBP-I) was assessed by Western blotting. Before supplementation with ROL, RE were lower in RCS rats. After ROL supplementation, esters increased and reached values that were similar in the two strains, but the increase, expressed relative to the initial value, was higher in RCS rats. The uptake of ROL and the level of CRBP-I were greater in RCS rats. Our results provide evidence of a functional retinol esterifying enzyme in cultured RCS RPE cells and suggest that CRBP-I could play a role in the uptake and esterification of ROL in the RPE cells.

The effect of intravitreal injections of DMTU (dimethylthiourea) and SOD (superoxide dismutase), two free radical scavengers, was evaluated in a rat model of retinal ischemia induced by elevated intraocular pressure. The drugs were administered just before or just after a 60 min ischemia. At days 2 and 7 after reperfusion, retinal recovery was evaluated by electroretinography. At day 7, layer thicknesses and cell rows were measured from histologic sections of paraffin-embedded retinas. In the vehicle-treated control group, we observed a decrease in the inner retinal layers and b-wave amplitude impairment. SOD injection (6 units/eye) protected the retina from ischemia/reperfusion injury. At day 2 after reperfusion, electroretinographic recovery was more efficient when SOD was administered just after ischemia (99%) than after pretreatment with SOD (81%) (p<0.03). In the DMTU-treated group (75 microg/eye), only the pretreatment induced significant electrophysiologic (40%) (p<0.001) and morphologic recovery.

Purpose: To investigate the functional protective effect of a synthetic (dimethylthiourea, DMTU) and a natural antioxidant (Ginkgo biloba extract, EGb 761) against light-induced retinal degeneration., Methods: Wistar rats were exposed for 24 hours to 1700-lux light after treatment with DMTU or EGb 761. Electroretinograms were recorded before and on day (D)1, D3, D8, D15, D22, and D29 after light exposure. The b-wave amplitude was plotted against log L (ganzfeld luminance), providing the b-wave sensitivity curve. The Naka-Rushton function fitted to the sensitivity curve enabled derivation of the parameters Bmax (saturated amplitude) and K (luminance-inducing Bmax/2). In addition, rats from each group were killed for retinal morphometric analyses., Results: In the untreated group, light exposure caused collapse of the b-wave sensitivity curves. Bmax was reduced by 51% at D1 without subsequent recovery. K increased temporarily, reverting to normal values 8 days later. The outer nuclear layer thicknesses decreased markedly in the superior retina. In the treated groups, light exposure had a weaker effect on sensitivity curves. The values of Bmax were not significantly different from those in the unexposed-untreated group, although K increased temporarily. Retinal morphometry was preserved., Conclusions: Dimethylthiourea and EGb 761 afford functional protection against light-induced retinal damage.

Multiple forms of phosphodiesterase have been reported in many tissues. Phosphodiesterase 6, a cGMP-specific phosphodiesterase, is described as a photoreceptor cell-specific phosphodiesterase. Phosphodiesterase 6 is known to play a crucial role in visual function. A novel phosphodiesterase inhibitor, GF248 (5["(propoxy),7'(4-morpholino)-phenacyl],[1-methyl-3 propyl]pyrazolo[4,3d]pyrimidin-7-one), has been described to be a very potent cGMP-specific phosphodiesterase inhibitor. In the present study, we compared the potency of GF248 and other known cGMP-specific phosphodiesterase inhibitors on phosphodiesterase 5 and phosphodiesterase 6. GF248 displayed an IC50 of 2 and 5 nM for phosphodiesterase 5 and phosphodiesterase 6, respectively. Thereafter, we assessed the effect of GF248 on retinal function, using an ex vivo model of isolated retina electroretinogram recording. Exposure of retina to GF248 resulted in a dose-dependent decrease in electroretinogram amplitude (PIII and b-waves), with no marked modification of PIII and b-wave implicit time. Among other phosphodiesterase inhibitors, DMPPO (1,3-dimethyl-6-(2-propoxy-5-methanesulfonylamidophenyl)pyrazol ol[3,4d]-pyrimidin-4-(5H)-one) and dipyridamole, cGMP-specific phosphodiesterase inhibitors, and IBMQ (1-isobutyl-3-methylimidazol[1,5a]quinoxalin-4-(5H)one), a nonselective phosphodiesterase inhibitor, altered retinal function but less potently than GF248, consistent with their in vitro phosphodiesterase 6 inhibition. Phosphodiesterase 3- and phosphodiesterase 4-selective inhibitors, cilostamide and rolipram, respectively, did not affect retinal function at 10 micromol l(-1). Our conclusion from these data is that GF248, a potent phosphodiesterase 6 inhibitor, could interfere with visual transduction by cGMP accumulation.

Purpose: ERG responses were measured as a function of Ganzfeld luminance to evaluate functional damage induced by light on rat retinas., Methods: Wistar rats were exposed to a fluorescent light of 1700 lux for 12 h, 24 h, 48 h and 72 h. We recorded ERGs before and one night after exposure, then 3, 8, 15, 22 and 29 days later. The b- and PIII-wave amplitudes were plotted against luminance for each group at each recovery time., Results: The retinal damage induced by a pupillary illuminance of 1700 lux ranged from low to severe as exposure duration increased from 12 h to 72 h, respectively. We observed an effect immediately after light exposure but no improvement during the recovery period. The b-wave amplitude was reduced by 40, 60, 80 and 90 percent after 12, 24, 48 and 72 h of light exposure, respectively; the PIII-wave amplitude was reduced by 30, 40, 70 and 90 percent after these respective exposures. The Ganzfeld luminance eliciting a 50 microV b-wave amplitude increased significantly with exposure duration, but the luminance eliciting the maximal b-wave amplitude was not dependent on this duration. Hence we suggest that the ERG decrease is due to a reduction in photoreceptor number., Conclusions: We present a full analysis of the electrophysiological parameters recorded from light-exposed or non-exposed rats. This model is a useful tool to study in vivo retinal degeneration.

Electroretinographic exploration is an effective approach to evaluate retinal function. In order to investigate physiopathological mechanisms and evaluate potentially protective therapies for retinal ischemia, we developed three experimental models: the first two on isolated retina, with ischemia induced by either stopping perfusion or clamping the ophthalmic artery, and the third, in vivo, with ischemia induced by ocular hypertonia. Since free radicals are implicated in the formation of post-ischemic lesions, we evaluated the protective effects of drugs known to be free radical scavengers and of an immunomediator antagonist, an anti-PAF (platelet activating factor) agent.

The alkaloid vincristine displays considerable toxicity, particularly for the retina. This type of retinopathy being an inflammatory disease, we measured the effects of a new hetrazepine platelet activating factor antagonist, BN 50730, on a vincristine-induced retinopathy in the rat. Retinal impairments were established by recording several parameters of the electroretinogram obtained from isolated retina. Our results indicate that 1) the increase in PIII duration induced by vincristine is significantly reduced by BN 50730 administration 2) the decrease in the amplitude of the PIII/b wave ratio caused by vincristine is partially inhibited by treatment with BN 50730. These experiments suggest that platelet activating factor is implicated in vincristine retinopathy and demonstrate the therapeutic effect of a specific antagonist of the mediator.

Several investigations have recently shown that the retina is very sensitive to oxygenated free radicals (O2-, OH.) at the origin of the membrane phospholipids peroxidation. Peroxy radical (ROO.) release is responsible for the induction of electrophysiological disturbances leading to retinopathy development. As Ginkgo biloba extract (EGb 761, IPSEN, France) was reported to scavenge primary (O2-, OH.) and secondary (ROO.) free radicals, we evaluated its antioxidant effect on retinas of albino rats submitted to different types of aggressors. On isolated rat retina, EGb 761 given orally significantly protected against lipoperoxidation induced by a mixture of ferrous sulfate and sodium ascorbate added to the perfusion solution. With EGb 761, the decrease of the b-wave ERG amplitude was less pronounced and the retina survival was increased. EGb 761 was also effective against ischaemia-reperfusion disorders due to occlusion of the central retinal artery or by intraocular hypertony. Like other antioxidants such as superoxide dismutase tested on these models, EGb 761 significantly attenuated, according to a dose-response effect, the free-radical injury. EGb 761 reduces the decrease of the b-wave amplitude, the oedema, necrosis and ion homeostasis disturbances. Xenobiotics are also responsible for the retinotoxicity partly due to free radicals and PAF release. We noted an EGb 761 dose-dependent protective effect against acute and chronic chloroquine toxicity to the retina. The deleterious effect of chloroquine was characterized by a delayed b-wave and an asymmetry of the signal with slow declining b-wave. After EGb 761 treatment, the ERG aspect was partially normal. In conclusion, EGb 761, by its general free-radical scavenger properties, is an antioxidant that inhibits or reduces the functional and morphological retina impairments observed after lipoperoxide release.

Platelet-activating factor (PAF) has been shown to alter the trans-retinal potential recorded from light-stimulated isolated retina. In the present study, we investigated the effect of cholera and pertussis toxins on PAF-induced impairment of the electroretinogram (ERG). Administered alone, 2 x 10(-7) M PAF induced a very marked and rapid drop in the b-wave amplitude. When 75 micrograms/l of cholera toxin was coadministered with PAF in the perfusion solution, no b-wave drop was observed, suggesting that the effect of PAF on retinal function was mediated by GTP-binding protein (G protein). Similarly, a low dose of pertussis toxin (5 micrograms/l) was sufficient to antagonize the action of PAF on the ERG. Our results suggest that the irreversible and deleterious effect of PAF on ERG is mediated by a G protein mechanism, located in the neural retina.

Chloroquine retinopathy is a severe toxic retinal impairment which may result in loss of vision by alterations of the retinal pigment epithelium and photoreceptors. Currently, there is no specific treatment for this retinopathy. Platelet-activating factor (PAF) is known to modulate retinal function and is one of the major immunomediators of the retina. In order to test the possible involvement of PAF in chloroquine-induced retinopathy and the effectiveness of PAF antagonists in the prevention of this condition, we investigated the effects of BN 50730, a specific PAF antagonist, on the electroretinogram (ERG) of the isolated rat retina exposed to chloroquine. When retinas from normal rats were perfused with chloroquine (10(-6) M), a marked and rapid decrease in b-wave amplitude was observed. In contrast, chloroquine had no effect on the b-wave of the retina isolated from animals pretreated with the PAF antagonist BN 50730 (30 mg/kg/day, i.p., for 5 days). The results obtained indicate that (i) chloroquine is a toxic drug for retinal function, (ii) PAF plays a key role in the mediation of chloroquine retinopathy and (iii) PAF antagonists may constitute valuable agents for the treatment of this retinal impairment.

The various hardware components of this scanning system, a mechanical split-beam scanner with its photomultiplier measuring slit, minimum analog electronics and an important digital part (computer, interface), are intimately interwoven by an elaborate software. A high degree of automation and sophistication is thus reached. On the one hand, all the photoelectrons coming from each light pulse are integrated. The result, after conversion to digital form enters the computer; a high precision is thus reached: 10(-5) absorbance unit (A.U.) under very favorable conditions, and 10(-4) A.U. for conventional sedimentation studies. On the other hand, during its motion along the image, the precise slit position is permanently known by the system: the sector image is thus segmented into precisely defined zones, inside which the average absorbancies are determined, punched, plotted and printed. New and very useful utilizations and improvements of analytical ultracentrifugation are now possible: the fixed radius mode, permits precise s determination by following the radial dilution; s can be precisely measured by time difference curves even when the solution absorbancy is comparable to the base line variations (0.01 A.U.); round trip analyses permit very long analyses without the absorbancies distributions distorsions due to very long one-way scans during sedimentations runs; reductions of the effect of the stray light by a factor of ten. This system has been in routine use for four years.

The specificity of the effects of platelet-activating factor (PAF) on the electrophysiology of the retina suggests the existence of specific sites for PAF in this tissue. In this study, we report the presence of tritiated PAF ([3H]PAF) specific binding sites in membrane preparations of the retina of albino rats. The binding of [3H]PAF was saturable, specific, time-dependent and reversible. Scatchard analysis of the data revealed that the high-affinity retinal binding site possessed a Kd of 2.9 +/- 0.4 nM and Bmax of 0.85 +/- 0.16 pmol/mg protein. These values are comparable with those found for the membranous PAF receptor sites in platelets, neutrophils, lung tissue and brain. We have recently reported that PAF dose dependently modulates the b-wave of the electroretinogram (ERG) obtained from the isolated rat retina. The results of the present study suggest that such PAF-induced disturbances of the ERG may be mediated via specific receptors located in the retina.