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P. Zhang, J. M. Randazzo, K. Blessing, M. F. Lou, P. F. Kador; Comparison of Signaling Between Diabetic Rat Lenses and Rat Lenses Cultured in vitro With High Glucose. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1453.
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© ARVO (1962-2015); The Authors (2016-present)
In vitro lens culture studies are often conducted as an alternative to the use of in vivo animals for elucidating the progression of cataract formation. This technique has especially been used to investigate the progression of sugar cataract formation. The purpose of this study was to compare signaling changes in rat lenses cultured in vitro in high glucose to changes observed in lenses from diabetic rats.
Lenses from young (100 g) Sprague Dawley rats were cultured for up to 48 hrs in TC-199-bicarbonate buffer containing either 30 mM fructose (control) or 30 mM glucose with/without 10 µM of the AR inhibitors (ARI) AL1576 or tolrestat. Diabetes was induced in 100 g rats with tail vein injection of 75 mg/kg streptozotocin and divided into groups receiving either regular rat chow (untreated diabetic group) or groups receiving chow containing either AL1576 (0.0125%) or tolrestat (0.5%). The rats were maintained for 10 weeks. Non-diabetic, age-matched rats were used as controls. GSH was spectrophotometrically measured using the DTNB method. Western blots were conducted using commercially available antibodies basic-FGF, IGF, TGFβ, P-Akt, P-c-Raf, P-SAPK/JNK, and P-Erk1/2.
Slight cortical opacities were observed in lenses cultured in high glucose for 48 hrs and mature cataracts were observed in all untreated diabetic rats. Lens changes were reduced in both in vitro and in vivo conditions by treatment with ARIs. Incubation of the lenses in high glucose media for 48 hrs resulted in reduced GSH levels; however, this reduction was less in the lenses treated with AR inhibitors. Similarly, GSH level in lenses from untreated diabetic rats was reduced and this reduction was less in the ARI treated groups. Increased basic-FGF, IGF, TGFβ and increased transduction pathways of P-Erk1/2, P-SAPK/JNK and P-Akt were similarly observed in lenses incubated in high glucose media and in lenses from untreated diabetic rats. An exception is P-c-Raf which decreased in untreated diabetic lenses but increased in in vitro cultured lenses. In all in vivo and in vitro cases, ARI treatment moved signaling toward control levels.
Both in vitro and in vivo studies implicate the accumulation of sorbitol in signaling changes. The P-c-Raf results suggest that caution must be used in interpreting stress signaling changes with in vitro changes perhaps only representing initial or early lens changes.
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