Glaucoma is the second leading cause of blindness worldwide, often associated with elevated intraocular pressure. Connective tissue growth factor (CTGF) is a mediator of pathological effects in the trabecular meshwork (TM) and Schlemm’s canal (SC). A novel, causative therapeutic concept which involves the intracameral delivery of small interfering RNA against CTGF is proposed. Layer-by-layer coated nanoparticles of 200–260 nm with a final layer of hyaluronan (HA) are developed. The HA-coating should provide the nanoparticles sufficient mobility in the extracellular matrix and allow for binding to TM and SC cells via CD44. By screening primary TM and SC cells in vitro, in vivo, and ex vivo, the validity of the concept is confirmed. CD44 expression is elevated in glaucomatous versus healthy cells by about two-to sixfold. CD44 is significantly involved in the cellular uptake of HA-coated nanoparticles. Ex vivo organ culture of porcine, murine, and human eyes demonstrates up to threefold higher accumulation of HA compared to control nanoparticles and much better penetration into the target tissue. Gene silencing in primary human TM cells results in a significant reduction of CTGF expression. Thus, HA-coated nanoparticles combined with RNA interference may provide a potential strategy for glaucoma therapy.

Nanotechnology has been proven to be increasingly compatible with pharmacological and biomedical applications. Therefore, we evaluated the biological interactions of single-wall carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG). For this purpose, we analyzed biochemical, histological, behavioral and biodistribution parameters to understand how this material behaves in vitro and in vivo using the fish Danio rerio (zebrafish) as a biological model. The in vitro results for fish brain homogenates indicated that SWNT-PEG had an effect on lipid peroxidation and GSH (reduced glutathione) content. However, after intraperitoneal exposure, SWNT-PEG proved to be less biocompatible and formed aggregates, suggesting that the PEG used for the nanoparticle functionalization was of an inappropriate size for maintaining product stability in a biological environment. This problem with functionalization may have contributed to the low or practically absent biodistribution of SWNT-PEG in zebrafish tissues, as verified by Raman spectroscopy. There was an accumulation of material in the abdominal cavity that led to inflammation and behavioral disturbances, as evaluated by a histological analysis and an open field test, respectively. These results provide evidence of a lack of biocompatibility of SWNTs modified with short chain PEGs, which leads to the accumulation of the material, tissue damage and behavioral alterations in the tested subjects.

Zusammenfassung Hitzegeschädigte Proteine mit einem hohen Gehalt an Fruktoselysin, dem wichtigsten Reaktionsprodukt von Lysin mit Glukose oder Laktose, wurden zusammen mit Darminhalt von Ratten inkubiert. Als Kontrollen dienten Ansätze, die zur Abtötung der Mikroorganismen mit Sublimat versetzt waren. Wie die Versuche ergaben, sind Mikroorganismen aus dem Verdauungstrakt befähigt, das fur höhere Tiere nicht spaltbare und nicht resorbierbare Fruk‐toselysin abzubauen. Wir danken der Deutschen Forschungsgemeinschaft für Unterstützung der Versuche. Summary Degradation of fructoselysine by the intestinal flora Heat damaged proteins with high contents of fructoselysine were incubated with gut contents of rats. Controls has been mixed before incubation with sublimate in order to stop the action of microorganisms. Fructoselysine is neither utilized by the animal nor absorbed as an intact molecule. Our results show however, that fructoselysine was deaminated by the microorganisms of the rats intestinal tract. Résumé Dégradation de la fructoselysine par la flore intestinale On incube avec le contenu intestinal de rats, des protéines dénaturées à la chaleur, à forte teneur en fructoselysine, qui est le produit de réaction le plus important de la lysine avec le glucose ou le lactose. Comme contrôles, on emploie les préparations qu'on a combinées avec du sublimé pour détruire les microorganismes. Comme les résultats l'ont montré, les microorganismes du tractus digestif sont capables de dégrader la fructoselysine ne pouvant être scindée, ni résorbée par les animaux supérieurs. Resumen Desintegración de la fructosalisina mediante la flora intestinal Junto con contenido entérico de ratas se incubaron proteínas, dañadas por el calor, con uncontenido elevado en fructosalisina, el producta de reacción más importante de la lisina con la glucosa o lactosa. Como testigos se utilizaron siembras que se habian tratado con sublimado para matar los micro‐organismos. Como prueban estos ensayos, los microbios del tracto digestivo son capaces de desintegrar la fructosalisina, que los animales superiores no escinden ni resorben.

Carbon nanotubes (CNT) are promising materials for biomedical applications, especially in the field of neuroscience; therefore, it is essential to evaluate the neurotoxicity of these nanomaterials. The present work assessed the effects of single-walled CNT functionalized with polyethylene glycol (SWCNT-PEG) on the consolidation and retrieval of contextual fear memory in rats and on oxidative stress parameters in the hippocampus. SWCNT-PEG were dispersed in water at concentrations of 0.5, 1.0, and 2.1 mg/mL and infused into the rat hippocampus. The infusion was completed immediately after training and 30 min before testing of a contextual fear conditioning task, resulting in exposure times of 24 h and 30 min, respectively. The results showed that a short exposure to SWCNT-PEG impaired fear memory retrieval and caused lipid peroxidation in the hippocampus. This response was transient and overcome by the mobilization of antioxidant defenses at 24 h. These effects occurred at low and intermediate but not high concentration of SWCNT-PEG, suggesting that the observed biological response may be related to the concentration-dependent increase in particle size in SWCNT-PEG dispersions.

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Carbon nanotubes are promising nanomaterials for the diagnosis and treatment of brain disorders. However, the ability of these nanomaterials to cross cell membranes and interact with neural cells brings the need for the assessment of their potential adverse effects on the nervous system. This study aimed to investigate the biopersistence of single-walled carbon nanotubes functionalized with polyethylene glycol (SWCNT-PEG) directly infused into the rat hippocampus. Contextual fear conditioning, Y-maze and open field tasks were performed to evaluate the effects of SWCNT-PEG on memory and locomotor activity. The effects of SWCNT-PEG on oxidative stress and morphology of the hippocampus were assessed 1 and 7 days after infusion of the dispersions at 0.5, 1.0 and 2.1 mg/mL. Raman analysis of the hippocampal homogenates indicates the biopersistence of SWCNT-PEG in the hippocampus 7 days post-injection. The infusion of the dispersions had no effect on the acquisition or persistence of the contextual fear memory; likewise, the spatial recognition memory and locomotor activity were not affected by SWCNT-PEG. Histological examination revealed no remarkable morphological alterations after nanomaterial exposure. One day after the infusion, SWCNT-PEG dispersions at 0.5 and 1.0 mg/mL were able to decrease total antioxidant capacity without modifying the levels of reactive oxygen species or lipid hydroperoxides in the hippocampus. Moreover, SWCNT-PEG dispersions at all concentrations induced antioxidant defenses and reduced reactive oxygen species production in the hippocampus at 7 days post-injection. In this work, we found a time-dependent change in antioxidant defenses after the exposure to SWCNT-PEG. We hypothesized that the persistence of the nanomaterial in the tissue can induce an antioxidant response that might have provided resistance to an initial insult. Such antioxidant delayed response may constitute an adaptive response to the biopersistence of SWCNT-PEG in the hippocampus.

Norrin is a secreted signaling molecule activating the Wnt/β-catenin pathway. Since Norrin protects retinal neurons from experimental acute injury, we were interested to learn if Norrin attenuates chronic damage of retinal ganglion cells (RGC) and their axons in a mouse model of glaucoma. Transgenic mice overexpressing Norrin in the retina (Pax6-Norrin) were generated and crossed with DBA/2J mice with hereditary glaucoma and optic nerve axonal degeneration. One-year old DBA/2J/Pax6-Norrin animals had significantly more surviving optic nerve axons than their DBA/2J littermates. The protective effect correlated with an increase in insulin-like growth factor (IGF)-1 mRNA and an enhanced Akt phosphorylation in DBA/2J/Pax6-Norrin mice. Both mouse strains developed an increase in intraocular pressure during the second half of the first year and marked degenerative changes in chamber angle, ciliary body and iris structure. The degenerations were slightly attenuated in the chamber angle of DBA/2J/Pax6-Norrin mice, which showed a β-catenin increase in the trabecular meshwork. We conclude that high levels of Norrin and the subsequent constitutive activation of Wnt/β-catenin signaling in RGC protect from glaucomatous axonal damage via IGF-1 causing increased activity of PI3K-Akt signaling. Our results identify components of a protective signaling network preventing degeneration of optic nerve axons in glaucoma.

High amounts of TGF-β in the eye cause a substantial reduction in the activity of Wnt/β-catenin signaling. This effect is inhibited in the presence of high amounts of Norrin, which further induce the expression of SMAD7 to inhibit TGF-β signaling.