Primary glia expressing the G93A-SOD1 mutation present a neuroinflammatory phenotype and provide a cellular system for studies of glial inflammation

Detailed study of glial inflammation has been hindered by lack of cell culture systems that spontaneously demonstrate the "neuroinflammatory phenotype". Mice expressing a glycine → alanine substitution in cytosolic Cu, Zn-superoxide dismutase (G93A-SOD1) associated with familial amyotrophic lateral sclerosis (ALS) demonstrate age-dependent neuroinflammation associated with broad-spectrum cytokine, eicosanoid and oxidant production. In order to more precisely study the cellular mechanisms underlying glial activation in the G93A-SOD1 mouse, primary astrocytes were cultured from 7 day mouse neonates. At this age, G93A-SOD1 mice demonstrated no in vivo hallmarks of neuroinflammation. Nonetheless astrocytes cultured from G93A-SOD1 (but not wild-type human SOD1-expressing) transgenic mouse pups demonstrated a significant elevation in either the basal or the tumor necrosis alpha (TNFα)-stimulated levels of proinflammatory eicosanoids prostaglandin E(2 )(PGE(2)) and leukotriene B(4 )(LTB(4)); inducible nitric oxide synthase (iNOS) and •NO (indexed by nitrite release into the culture medium); and protein carbonyl products. Specific cytokine- and TNFα death-receptor-associated components were similarly upregulated in cultured G93A-SOD1 cells as assessed by multiprobe ribonuclease protection assays (RPAs) for their mRNA transcripts. Thus, endogenous glial expression of G93A-SOD1 produces a metastable condition in which glia are more prone to enter an activated neuroinflammatory state associated with broad-spectrum increased production of paracrine-acting substances. These findings support a role for active glial involvement in ALS and may provide a useful cell culture tool for the study of glial inflammation

Chemical versus Pharmacological Actions of Nutraceutical Phytochemicals: Antioxidant and Anti Inflammatory Modalities

Article on chemical versus pharmacological actions of nutraceutical phytochemicals and antioxidant and anti inflammatory modalities

Catalytic Nanoceria Are Preferentially Retained in the Rat Retina and Are Not Cytotoxic after Intravitreal Injection

Cerium oxide nanoparticles (nanoceria) possess catalytic and regenerative radical scavenging activities. The ability of nanoceria to maintain cellular redox balance makes them ideal candidates for treatment of retinal diseases whose development is tightly associated with oxidative damage. We have demonstrated that our stable water-dispersed nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in vldlr mutant mice. The objectives of the current study were to determine the temporal and spatial distributions of nanoceria after a single intravitreal injection, and to determine if nanoceria had any toxic effects in healthy rat retinas. Using inductively-coupled plasma mass spectrometry (ICP-MS), we discovered that nanoceria were rapidly taken up by the retina and were preferentially retained in this tissue even after 120 days. We also did not observe any acute or long-term negative effects of nanoceria on retinal function or cytoarchitecture even after this long-term exposure. Because nanoceria are effective at low dosages, nontoxic and are retained in the retina for extended periods, we conclude that nanoceria are promising ophthalmic therapeutics for treating retinal diseases known to involve oxidative stress in their pathogeneses

Defining the Catalytic Activity of Nanoceria in the P23H-1 Rat, a Photoreceptor Degeneration Model

<div><p>Purpose</p><p>Inorganic catalytic nanoceria or cerium oxide nanoparticles (CeNPs) are bona fide antioxidants that possess regenerative radical scavenging activities <i>in vitro</i>. Previously, we demonstrated that CeNPs had neuroprotective and anti-angiogenic properties in rodent retinal degeneration and neovascularization models. However, the cellular mechanisms and duration of the catalytic activity of CeNPs in preventing photoreceptor cell loss are still unknown. In this study, we sought to answer these questions using the P23H-1 rat, an autosomal dominant retinitis pigmentosa (adRP) model.</p><p>Methods</p><p>A single dose of either saline or CeNPs was delivered intravitreally into the eyes of P23H-1 rats at 2–3 weeks of age. Retinal functions were examined at 3 to 7 weeks post injection. We quantified retinal proteins by Western blot analyses and counted the number of apoptotic (TUNEL+) profiles in the outer nuclear layer (ONL) of retinal sections. We measured free 8-isoprostanes to quantify lipid peroxidation in retinal tissues.</p><p>Results</p><p>We observed increased rod and cone cell functions up to three weeks post injection. Apoptotic cells were reduced by 46%, 56%, 21%, and 24% at 3, 7, 14, 21 days, respectively, after CeNPs injection compared to saline. Additionally, reduction of lipid peroxidation in the retinas of CeNPs-treated vs saline-treated animals was detected 14 days post injection.</p><p>Conclusions</p><p>We validated that CeNPs were effective in delaying loss of photoreceptor cell function in an adRP rat model. This represents the fourth rodent retinal disease model that shows delay in disease progression after a single application of CeNPs. We further demonstrated that CeNPs slowed the rate of photoreceptor cell death. We deduced that the catalytic activity of CeNPs <i>in vivo</i> in this rat model to be undiminished for at least 7 days and then declined over the next 14 days after CeNPs administration.</p></div

A single application of CeNPs at P15 reduced the number of apoptotic death of photoreceptor cells for at least 21 days.

<p>(A-B) Representative photomicrographs of retinal sections from 3 dpi group with examples of TUNEL+ profiles (green). Nuclei in the ONL and INL are labeled blue. Rod outer segments are labeled red with Rhodopsin, 1D4, antibody. CeNPs-treated animals have significantly fewer TUNEL+ profiles in the ONL compared to saline-treated ones. (C) Quantification of TUNEL+ profiles in the ONL from 3, 7, 14, and 21 dpi groups. The reductions from 3 and 7 dpi groups of CeNPs injected animals were highly significant. Statistical summaries from these box plots are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121977#pone.0121977.t002" target="_blank">Table 2</a>. Abbreviations: dpi = days post injection, S = Saline, C = CeNPs, *p<0.05.</p

Statistical Summaries of TUNEL+ profiles in the ONL of Retinal Sections from CeNPs and Saline Treated P23H-1 Rats.

<p>Animals were injected at P15 and eyes were harvested at 3, 7, 14, and 21 days post injection. Abbreviations: Sal = Saline, CNP = CeNP, dpi = days post injection.</p><p>Statistical Summaries of TUNEL+ profiles in the ONL of Retinal Sections from CeNPs and Saline Treated P23H-1 Rats.</p

Morphometric analysis of ONL thickness in CeNPs- and saline- treated P23H-1 rats.

<p>One μl of 1 mM CeNPs (172 ng) or saline was delivered to each eye of the animals at P23 and eyes were harvested at 28 dpi; three eyes from 3 individuals were examined per treatment group. (A-D) Representative photomicrographs of H&E stained retinal sections from wildtype, Sprague Dawley (SD) or P23H-1 animals uninjected or treated with either saline or CeNPs. Similar regions were shown: 1 mm from the ONH in the inferior region. (E) shows quantification of the ONL thickness measurements. The overall ONL thickness was higher in CeNPs-treated than in saline-treated animals although the increases were not statistically significant across many of the regions. INL = inner nuclear layer, ONL = outer nuclear layer, I/OS = rod inner/outer segment, ONH = optic nerve head. *P<0.05.</p

Quantification of lipid peroxidation in retinal samples of P23H-1 rats.

<p>(A) Retinas of 28-day old P23H-1 rats had elevated levels of 8-isoprostanes compared to age-matched wildtype, SD controls. N = 4, #P<0.01. (B) A single CeNPs application at P15 reduced the level of 8-isoprostanes 14 days later in the retinas of P23H-1 rats. N = 5–6, *P<0.05. Abbreviations: SD = Sprague Dawley, Sal = Saline, CNP = CeNPs.</p