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Reprogramming of toll-like receptor 4 (TLR4) by brief ischemia or lipopolysacharide (LPS) contributes to superintending tolerance against destructive ischemia in brain. However, beneficial roles of TLR4 signaling in ischemic retina are not well known. This study demonstrated that preconditioning with LPS 48 h prior to the retinal ischemia prevents the cellular damage in morphology with hematoxylin and eosin (H&E) staining and functions of retina with electroretinogram (ERG), while post-ischemia treatment deteriorated it. The preventive effects of LPS preconditioning showed the cell type-specificity of retinal cells. There was complete rescue of ganglion cells, partial rescue of bipolar and photoreceptor cells or no rescue of amacrine cells, respectively. LPS treatment caused the proliferation and migration of retinal microglia and its preconditioning prevented the ischemia-induced microglial activation. Preventive actions from cell damages following LPS preconditioning prior to retinal ischemia were abolished in TLR4 knock-out mice, and by pre-treatments with anti-TLR4 antibody or minocycline, a microglia inhibitor, which themselves had no effects on the retinal ischemia-induced damages or microglia activation. Thus, this study revealed that TLR4 mediates the LPS preconditioning-induced preventive effects through microglial activation in the retinal ischemia model.
Ischemia in the central nervous system including retina is one of the most well-known pathophysiological condition, which leads to extensive neuronal damages and functional disorders by triggering diverse types of self-reinforcing destructive mechanisms, such as necrosis and apoptosis (Lipton 1999; Bernstein et al. 2003; Danton and Dietrich 2003; Osborne et al. 2004; Ueda and Fujita 2004; Arumugam et al. 2006; Kaur et al. 2008; Lakhan et al. 2009; Neroev et al. 2010; Iadecola and Anrather 2011). These neuronal damages in retina are caused by ischemia-induced activation of detrimental cascades including up-regulation and secretion of injury-related cytokines derived from retinal glial cells (Neufeld et al. 2002; Osborne et al. 2004; Langmann 2007; Kaur et al. 2008; Neroev et al. 2010; Cervia and Casini 2012). Among these deleterious pathways, toll-like receptor 4 (TLR4) functions as a key component to mediate ischemic damages in the brain and retina through generation of several cytotoxic mediators, and mice lacking functional TLR4 shows partial neuroprotection against brain ischemia (Cao et al. 2007; Caso et al. 2007; Hua et al. 2007; Lehnardt et al. 2008; Marsh et al. 2009b; Dvoriantchikova et al. 2010; Hyakkoku et al. 2010; Ko et al. 2011; Wang et al. 2011). Similar cytotoxic effects are caused by the treatment with lipopolysaccharide (LPS), an endotoxin and known specific ligand for TLR4 (Guha and Mackman 2001; Sheng et al. 2003; Kawai and Akira 2007; Liang et al. 2007; Qin et al. 2007; Litvak et al. 2009; Jeong et al. 2010; Pang et al. 2012). However, it is also reported that the LPS preconditioning leads to robust neuroprotection against lethal cerebral ischemia through TLR4 signaling (Tasaki et al. 1997; Bastide et al. 2003; Rosenzweig et al. 2004; Marsh et al. 2009a, b; Vartanian and Stenzel-Poore 2010; Stevens et al. 2011; Vartanian et al. 2011). Although such contradictory cytotoxic and preventive roles of TLR4-mediated actions against ischemia may be in part attributed to the involvements of secondary produced glial cytokines (Obrenovitch 2008; Shpargel et al. 2008; Pradillo et al. 2009; Vartanian and Stenzel-Poore 2010; Wang et al. 2011; Chen et al. 2012), detailed and sequential machineries through glial cells remain elusive. One of difficulties in the study using cerebral ischemia model and systemic LPS treatment is ascribed to multiple parameters, including actions on peripheral immune cells, vascular endothelial cells, and brain glial cells. Even if using the intracerebroventricular administration of LPS, sequential analyses through glial cells seem to be difficult because of its regionally uneven brain distribution.
To answer this problem, we decided to use the retinal ischemia model, since it is very convenient, in terms of the simplicity of histological and functional studies (Fujita et al. 2009; Perlman 2009; Ueda et al. 2010, 2012; Halder et al. 2013). In addition, retinal cell layers are organized in proper orders, along with availability of biomarkers for specific cells (Sherry et al. 2006; Rhee et al. 2007; Buckingham et al. 2008; Masland 2012; Halder et al. 2013). This cell layer function is also evaluated by use of electroretinogram (Fujita et al. 2009; Perlman 2009; Halder et al. 2013). Most importantly, in the experiment using intravitreous injection, we can discuss the effects on all retinal cell layers as well as glial cells in closed and very small volume of space. In this study, we attempted to characterize the LPS-induced protection of retinal cells and its glial involvements in the retinal ischemia model.
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- Materials and methods
Retinal ischemia is associated with multiple sight-threatening disorders and blindness including macular degeneration, retinopathy and glaucoma, in which the event of functional damage and neuronal loss are mediated by a large array of injury-related signaling, such as excitatory neurotransmitter release, Ca2+ overload, formation of reactive oxygen species and free radicals, and secretion of potentially toxic mediators from glial cells, depending on the strength and duration of the ischemic insult (Osborne et al. 2004; Kaur et al. 2008; Hernandez et al. 2009; Neroev et al. 2010; Cervia and Casini 2012). Innate immunity system through TLR4 also contributes to this retinal ischemic damages (Dvoriantchikova et al. 2010), its beneficial roles have been reported in case with cerebral ischemia model, in which preconditioning with lower doses of LPS prevents the ischemia-induced brain damages through TLR4 signaling (Marsh et al. 2009a, b; Vartanian et al. 2011). In this study, we confirmed that the preconditioning of LPS significantly prevented the cell damages in all retinal layers after the ischemia. The best prevention in the evaluation with H&E histology and ERG was obtained when the preconditioning was given 48 h before the ischemic stress. The local intravitreous administration of LPS at as low as 0.1 and 1 μg significantly rescued the cellular loss and decrease in total thickness of ischemic retina at day 7, while there was a slow decline with 5 μg. As the LPS-induced preventive effect on GCL was maximal at 1 μg LPS, the decline with 5 μg seems to be attributed to its toxic effect, since it is reported that the activation of TLR4 by higher dose of LPS induces the robust production of inflammatory molecules through different cascades including myeloid differentiation factor (MyD)88-nuclear factor κB (NF-κB) pathway and MyD88-deficient cells failed to produce inflammatory cytokines in response to LPS (Palsson-McDermott and O'Neill 2004; Broad et al. 2007; Kawai and Akira 2007; Piao et al. 2009; Maitra et al. 2012). Furthermore, MyD88-dependent pathway is involved in an early response to LPS, followed by late activation of TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent cascade of TLR4 (Palsson-McDermott and O'Neill 2004). In contrast, lower dose of LPS is mainly associated with the activation of MyD88-independent interferon regulatory factor 3 (IRF3)-TRIF pathway of TLR4 signaling, leading to induction of anti-inflammatory molecules and type I interferons (IFNs), such as IFN-β and concomitant suppression of NF-κB-mediated signaling, though several reports suggested the initial induction of low-grade pro-inflammatory cytokines through MyD88-dependent pathway and other unknown mechanisms upon this LPS preconditioning (Marsh et al. 2009b; Vartanian and Stenzel-Poore 2010; Stevens et al. 2011; Vartanian et al. 2011). This suppression of MyD88-NF-κB pathway is caused by several negative regulators, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), including interleukin-1 receptor-associated kinase (IRAK)-M and tripartite motif family (TRIM)-30, which are produced by low-dose LPS-induced TLR4 signaling (Vartanian and Stenzel-Poore 2010; Stevens et al. 2011; Wang et al. 2011). Previous reports suggested the up-regulation downstream anti-inflammatory genes of IRF3-TRIF pathway and down-regulation of downstream inflammatory genes IL-6, IL-1β, COX-2, and TNFα of MyD88-NF-κB pathway in LPS-preconditioned cerebral ischemic mice (Stevens et al. 2011; Vartanian et al. 2011; Wang et al. 2011). We found that the downstream neuroprotective genes of IRF3-TRIF pathway IL1RN and IFIT1 as well as the suppressor genes of MyD88-NF-κB pathway SOCS 1 and SOCS 3 are increased by LPS treatment, and this up-regulation was blocked by pre-treatment with anti-TLR4 antibody. On the other hand, the ischemia-induced up-regulation of downstream injury genes TNFα, IL-1β, IL-6, COX-2, and MCP-1 of MyD88-NF-κB pathway was markedly inhibited by LPS preconditioning, being consistent with the previous study against brain ischemia (Vartanian et al. 2011). Thus, low-dose LPS preconditioning causes the reprogramming of transcriptional response to TLR4 by enhancing the TRIF signaling and suppressing the NF-κB-induced production of cytotoxic molecules. However, detailed studies of anti-TLR4 antibody treatment or in TLR4−/− mice of LPS preconditioning-induced alternation of protective and injury genes upon ischemia, and exhaustive gene expression profile by use of microarray technique should be the next subjects.
Photosignal processing in the retina is mediated by sequential activation of three cell layers, ONL including rod and corn cells, INL including bipolar, amacrine, and horizontal cells, and GCL including ganglion cells, and the damage of any cell layers causes crucial problems in the signal processing. Previous investigators described that ischemia-induced cytotoxic signaling causes the damages of all these retinal cells, while ganglion cells are more vulnerable to ischemic stress than others (Osborne et al. 2004; Uckermann et al. 2005; Langmann 2007; Jehle et al. 2008; Kaur et al. 2008; Neroev et al. 2010; Cervia and Casini 2012). In this study, ganglion cells were seriously damaged by the ischemia, while this damage was perfectly prevented by LPS preconditioning, being in a good contrast with the case of amacrine cells, whose damages were not at all prevented by LPS. As reported in the case with cerebral ischemia (Marsh et al. 2009a; Stevens et al. 2011; Vartanian et al. 2011; Wang et al. 2011), LPS preconditioning effects in the retinal model were abolished in TLR4−/− mice or by the intravitreous pre-treatment with anti-TLR4 antibody. It should be noted that the thickness of IPL and INL was partially, but significantly prevented when injected this antibody alone 30 min before ischemia, whereas the basal retinal ischemia-induced damages was not affected when treated with anti-TLR4 antibody alone 2 days before ischemia in vehicle-preconditioned WT ischemic mice. This difference may be explained by the possibility that anti-TLR4 antibody is degraded in vivo during the period of two days. Furthermore, TLR4−/− mice also showed the significant retinal protection underlying the recovery of IPL and INL against retinal ischemic damages, being consistent with cerebral ischemia, in which partial prevention is observed in TLR4−/− mice (Cao et al. 2007; Hyakkoku et al. 2010). In addition, mice that have been preconditioned showed a marked reduction in cytotoxic mediators including TNFα, iNOS, and COX-2 in the brain of TLR4−/− mice (Pradillo et al. 2009). Thus, no alteration of retinal ischemia-induced damages by long-term pre-treatment of anti-TLR4 antibody alone or partial protection by treatment of anti-TLR4 antibody immediately before ischemia and in TLR4−/− mice may be attributed to higher contribution of TLR4-independent toxicity.
Several reports suggested the expression of TLR4 on the cell surface in murine microglia in the brain, while astrocytes express it intracellularly (Bsibsi et al. 2002; Olson and Miller 2004; Jack et al. 2005; Marsh et al. 2009b). In this study, the double fluorescence immunohistochemical data showed that TLR4 is expressed in retinal microglia in the GCL and IPL, whereas 1 μg LPS treatment caused a migration of TLR4-positive microglia throughout the different retinal cell layers and increased its number at day 2 after injection. It should be noted that there was no change in morphology of LPS-treated microglia, while a marked hypertrophy in nucleus and processes of ischemia-treated microglia. This difference may suggest that retinal ischemia causes TLR4-independent toxicity to a high degree as well as a slight degree of TLR4-dependent toxicity, which was evidenced by the experiment using anti-TLR4 antibody.
On the other hand, pre-treatment with minocycline, a microglial inhibitor caused the blockade of LPS-induced mild activation and migration of retinal microglia via TLR4. We found that minocycline pre-treatment causes the complete blockade of LPS preconditioning-induced prevention of retinal ischemia-induced functional damages and survival of ganglion, bipolar, and photoreceptor cells. However, minocycline alone failed to protect the ischemic damages in retina, when it was treated 48 h before ischemia. This may be explained by the decrease in efficacy during long-term incubation, since the average half-life of minocycline is reported as 15 h (Xiao et al. 2012). Thus, it is suggested that LPS preconditioning-induced protection against ischemia is mediated by microglia. Microglia is highly activated by its robust change in morphology upon ischemia in brain and in retina, and subsequently causes tissue damages by releasing a multitude of noxious mediators, but minocycline reverses these effects (Mabuchi et al. 2000; Tikka et al. 2001; Lai and Todd 2006; Langmann 2007; Plane et al. 2010). In contrast, microglia have neuroprotective actions by producing anti-inflammatory factors including neurotrophins (Streit 2002; Denes et al. 2007; Lalancette-Hebert et al. 2007; Shpargel et al. 2008; Lambertsen et al. 2009; Ransohoff and Perry 2009; Chen et al. 2012). Retinal microglia is normally localized in the GCL and IPL in retina and is superficially exposed by intravitreal LPS administration. It is noted that LPS preconditioning did not affect the astrocyte activation with or without ischemia. Thus, the mild activation of microglia following LPS preconditioning may be responsible for retinal protection against ischemia.
In contrast, we found that post-treatment with 1 μg LPS, as applied in preconditioning experiments deteriorates the ischemia-induced cellular and functional damages in retina at day 4, followed by maximum damages at day 7 after the ischemic stress. LPS post-treatment-induced retinal damage was significantly reversed by treatment with anti-TLR4 antibody, suggesting that LPS post-ischemia treatment induces cytotoxic effects through TLR4 signaling. This LPS action is not attributed to the TLR4 reprogramming, because brief ischemia- or LPS preconditioning-induced TLR4 reprogramming provides neuroprotection against lethal ischemia (Marsh et al. 2009a; Pradillo et al. 2009; Vartanian et al. 2011). Taken together, this study indicates the LPS preconditioning caused functional and cell type-specific protection of retina against ischemic damages, whereas its post-ischemia treatment failed to protect the ischemic damages. This LPS preconditioning-induced retinal protection is mediated by TLR4 signaling in microglia.
In conclusion, LPS treatment 48 h before retinal ischemia prevented the functional damages of ischemic retina, whereas its post-ischemia treatment failed to protect the ischemic damages. LPS preconditioning completely rescued the ischemia-induced ganglion cell loss, along with partial recovery of bipolar and photoreceptor cell damages. This LPS-induced retinal protection against ischemia was mediated through microglial TLR4 signaling. Therefore, detailed mechanisms underlying LPS preconditioning caused protection could give us novel information for the discovery of new drugs to prevent the ischemic disorders in the central nervous system.