Retinal neuroinflammatory induced neuronal degeneration - Role of toll-like receptor-4 and relationship with gliosis.

The purpose of this study was to explore retina-intrinsic neuroinflammatory reactions, effects on neuronal survival, relationship with classic gliosis, and possible role of the toll-like receptor 4 (TLR4). To isolate the adult retina from the systemic immune system, a previously described large animal explant culture model was used in which full-thickness porcine retinal sheets can be kept in vitro for extended time periods. Explants were kept for 5 days in vitro (DIV) and were treated with either; lipopolysaccharide (LPS), a Toll-like receptor-4 (TLR4) inhibitor (CLI-095), LPS + CLI-095, or solvent vehicle throughout the culture period after which retinal sections were examined with hematoxylin and eosin staining and extensive immunohistochemistry. In addition, culture medium from all explant groups was assayed for a panel of cytokines at 2 and 5DIV. Compared with in vivo controls, vehicle controls (CT) as well as CLI-095 explants displayed moderate reduction of total thickness and number of retinal neurons with upregulation of glial fibrillary acidic protein (GFAP) throughout the Müller cells. In contrast, LPS and LPS + CLI-095 treated counterparts showed extensive overall thinning with widespread neuronal degeneration but only minimal signs of classical Müller cell gliosis (limited upregulation of GFAP and no downregulation of glutamine synthetase (GS). These specimens also displayed a significantly increased expression of galectin-3 and TGF-beta activated kinase 1 (TAK1). Multiplex proteomic analysis of culture medium at 2DIV revealed elevated levels of IL-1ß, IL-6, IL-4 and IL-12 in LPS-treated explants compared to CLI-095 and CT counterparts. LPS stimulation of the isolated adult retina results in substantial neuronal cell death despite only minimal signs of gliosis indicating a retina-intrinsic neuroinflammatory response directly related to the degenerative process. This response is characterized by early upregulation of several inflammatory related cytokines with subsequent upregulation of Galectin-3, TLR4 and TAK1. Pharmacological block of TLR4 does not attenuate neuronal loss indicating that LPS induced retinal degeneration is mediated by TLR4 independent neuroinflammatory pathways.

Acute tissue reactions, inner segment pathology, and effects of the antioxidant α1-microglobulin in an in vitro model of retinal detachment.

The purpose of this study was to explore acute tissue reactions, ultrastructural photoreceptor morphology with emphasis on inner segments, and the effect of antioxidant treatment in an in vitro model of rhegmatogenous retinal detachment (RRD). A previously described method of RRD simulation was used with adult retinal porcine explants kept free-floating in culture medium with or without treatment with the radical scavenger α1-microglobulin (A1M). Explants were examined at 5 time points from 1 to 24 h using transmission electron microscopy as well as quantitative real-time PCR (RT-PCR) to quantify gene expression of the cell stress marker heat shock protein 70 (Hsp70) and oxidative stress marker heme oxygenase (HO-1). The culture medium level of the cell damage marker lactate dehydrogenase (LDH) and oxidative stress DNA damage marker 8-Oxo-2'-deoxyguanosine (8-OHdG) was also assessed at each time point. We found that the levels of Hsp70 and LDH rapidly increased in both groups, and at 3 and 6 h, Hsp70 was significantly higher in A1M treated retinas. At 24 h, Hsp70 and LDH, as well as 8-OHdG were significantly lower compared with controls, whereas the tissue level of HO-1 was significantly higher. Progressive ultrastructural photoreceptor changes were seen in untreated control explants from 1 h and onwards including outer segment shortening and loss, disruption of organelles within the inner segments and loss of perikarya in the outer nuclear layer. Inner segment pathology was more rapid and extensive in rods compared with in cones. In A1M treated counterparts, damage to rod inner segment mitochondria was significantly higher after 1 h of culture, but after this time, no statistical difference was found. At 24 h, cone inner segment mitochondrial disruption was significantly higher in control retinas and the number of surviving perikarya lower. From our results, we conclude that retinal explants elicit acute cell stress reactions when placed in culture without physical support simulating a detached retina floating in the vitreous space. Photoreceptors rapidly display degenerative changes including extensive damage to inner segment mitochondria indicating loss of energy transduction as an early key event. A1M increases initial mitochondrial stress in the rods, however, subsequent pathology is attenuated by the treatment, highlighting the dynamics of protective as well as disruptive oxidative stress reactions in the detached retina.

Inflamed In Vitro Retina: Cytotoxic Neuroinflammation and Galectin-3 Expression.

BACKGROUND: Disease progression in retinal neurodegeneration is strongly correlated to immune cell activation, which may have either a neuroprotective or neurotoxic effect. Increased knowledge about the immune response profile and retinal neurodegeneration may lead to candidate targets for treatments. Therefore, we have used the explanted retina as a model to explore the immune response and expression of the immune modulator galectin-3 (Gal-3), induced by the cultivation per se and after additional immune stimulation with lipopolysaccharide (LPS), and how this correlates with retinal neurotoxicity. METHODS: Post-natal mouse retinas were cultured in a defined medium. One group was stimulated with LPS (100 ng/ml, 24 h). Retinal architecture, apoptotic cell death, and micro- and macroglial activity were studied at the time of cultivation (0 days in vitro (DIV)) and at 3, 4 and 7 DIV using morphological staining, biochemical- and immunohistochemical techniques. RESULTS: Our results show that sustained activation of macro- and microglia, characterized by no detectable cytokine release and limited expression of Gal-3, is not further inducing apoptosis additional to the axotomy-induced apoptosis in innermost nuclear layer. An elevated immune response was detected after LPS stimulation, as demonstrated primarily by release of immune mediators (i.e. interleukin 2 (IL-2), IL-6, KC/GRO (also known as CLCX1) and tumour necrosis factor-α (TNF-α)), increased numbers of microglia displaying morphologies of late activation stages as well as Gal-3 expression. This was accompanied with increased apoptosis in the two additional nuclear layers, and damage to retinal gross architecture. CONCLUSION: We demonstrate that an immune response characterized by sustained and increased release of cytokines, along with an increase in Gal-3 expression, is accompanied by significant increased neurotoxicity in the explanted retina. Further investigations using the current setting may lead to increased understanding on the mechanisms involved in neuronal loss in retinal neurodegenerations.

Silver and gold nanoparticles exposure to in vitro cultured retina--studies on nanoparticle internalization, apoptosis, oxidative stress, glial- and microglial activity.

The complex network of neuronal cells in the retina makes it a potential target of neuronal toxicity--a risk factor for visual loss. With growing use of nanoparticles (NPs) in commercial and medical applications, including ophthalmology, there is a need for reliable models for early prediction of NP toxicity in the eye and retina. Metal NPs, such as gold and silver, gain much of attention in the ophthalmology community due to their potential to cross the barriers of the eye. Here, NP uptake and signs of toxicity were investigated after exposure to 20 and 80 nm Ag- and AuNPs, using an in vitro tissue culture model of the mouse retina. The model offers long-term preservation of retinal cell types, numbers and morphology and is a controlled system for delivery of NPs, using serum-free defined culture medium. AgNO3-treatment was used as control for toxicity caused by silver ions. These end-points were studied; gross morphological organization, glial activity, microglial activity, level of apoptosis and oxidative stress, which are all well described as signs of insult to neural tissue. TEM analysis demonstrated cellular- and nuclear uptake of all NP types in all neuronal layers of the retina. Htx-eosin staining showed morphological disruption of the normal complex layered retinal structure, vacuole formation and pyknotic cells after exposure to all Ag- and AuNPs. Significantly higher numbers of apoptotic cells as well as an increased number of oxidative stressed cells demonstrated NP-related neuronal toxicity. NPs also caused increased glial staining and microglial cell activation, typical hallmarks of neural tissue insult. This study demonstrates that low concentrations of 20 and 80 nm sized Ag- and AuNPs have adverse effects on the retina, using an organotypic retina culture model. Our results motivate careful assessment of candidate NP, metallic or-non-metallic, to be used in neural systems for therapeutic approaches.