No abstract

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Mü ller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Mü ller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Mü ller cells, under H 2 O 2 and IL-1β damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H 2 O 2 and IL-1β, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNFα), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H 2 O 2 and IL-1β. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.

Purpose Human cord blood serum (CBS) is enriched with neurotrophins endowed of neuroprotective functions. Muller cells orchestrate retinal functioning, but their hyper‐activation can exacerbate retinal damage under oxidative stress, which typically occurs during neurodegenerative diseases. In this study we tested whether CBS could regulate human Muller cell activation under H2O2‐ and IL‐1□‐ induced damage. Methods Selected neurotrohpins (BDNF, TGF‐□, NGF, GDNF, EGF) were analysed in CBS (Human Magnetic Luminex Screening Assay). The human Muller cell line Moorfields/Institute of Ophtalmology‐Muller 1 (MIO‐M1) was obtained from the UCL Institute of Ophtalmology, London, UK1. MIO‐M1 were seeded in growth media enriched with standard FBS or CBS, both at 5%. After 24 h, MIO‐M1 were exposed to H2O2 and IL‐1□ for 24 h. CBS effects were compared to FBS control cells in terms of cell survival (MTT assay), activation (GFAP mRNA) and inflammation (TNF‐□ and IL‐6 mRNA). Results Median levels of neurotrophins in CBS were: BDNF 1.5 ng/ml, NGF 3.0 pg/ml, GDNF 1.5 pg/ml, TGF‐□ 36.3 pg/ml, EGF 820 pg/ml. MIO‐M1 pre‐cultured with CBS and exposed to H2O2, underwent a 50% decrease of GFAP mRNA in comparison to FBS control cells. CBS also mitigated the inflammatory activation, as shown by a significant 50% reduction of IL‐6 and TNF‐□ mRNA levels. The CBS ability to modulate the inflammatory pathway was confirmed by the significant decrease of IL‐6 and TNF‐□ genes (6‐ and 4‐ fold lower than FBS, respectively) in MIO‐M1 exposed to IL‐1□ (50 ng/ml). Conclusion CBS protects retinal Muller cells from damage under in vitro stress conditions. The main targets of this neuroprotecting stretegy are GFAP, marker of Muller cell activation, and the inflammatory cytokines, closely related to the neurodegenerative diseases. Therefore, CBS represents a promising therapeutic agent for the neurodegenerative diseases and further studies are necessary to validate this issue. Reference Limb GA et al. 2002, IOVS, 43; 864–869.