Systemic inflammation induced by lipopolysaccharide aggravates inherited retinal dystrophy

Retinal neurodegenerative diseases involve a scenario of inflammation and cell death that leads to morphological alterations and visual impairment. Non-ocular inflammatory processes could affect neurodegenerative retinal disorders and their progression, at least in part by activating microglial cells and releasing pro-inflammatory cytokines. Our purpose was to study the consequences of a systemic inflammatory process in the progression of retinal degeneration in P23H rats, a retinitis pigmentosa (RP) model. In order to induce a mild chronic systemic inflammation, we administered low doses of lipopolysaccharide (LPS) from age P20 to P60 to dystrophic P23H rats and healthy SD rats. Visual responsiveness was assessed by electroretinography (ERG). The morphological state of the retinas was analyzed by fluorescent immunohistochemistry (IHC), evaluating the number, morphology, and connectivity of different neuronal populations by means of cell type-specific markers. Microglia density, distribution, and degree of activation were evaluated by IHC and flow cytometry. The expression levels of inflammation- and apoptosis-related genes were analyzed by qRT-PCR arrays. Low-dose LPS administration did not induce significant functional or morphological changes in the retina of SD rats, although at the molecular level, we detected expression changes in genes related to apoptosis. Otherwise, systemic injection of LPS into P23H rats induced a further deterioration in the ERG response, with greater loss of photoreceptors and worsening of synaptic connectivity, accompanied by increasing numbers of microglial cells, which also showed a more intense activation state. Several inflammation- and apoptosis-related genes were upregulated. Our results indicate that chronic exacerbation of the inflammatory response in response to LPS accelerates neurodegeneration in dystrophic P23H rats, suggesting that in patients with ocular neurodegenerative diseases, peripheral damage, as a systemic infection or chronic inflammatory process, could accelerate disease progression, and should be taken into account in order to select an appropriate therapy to revert, block or slow-down the degenerative process.


Introduction
Retinal neurodegenerative diseases lead to morphological and functional impairment within a scenario of inflammation in which the three main cell death pathways (apoptosis, necrosis, and autophagy) converge [1][2][3][4] . Microglia activation and elevated levels of pro-inflammatory cytokines are a common feature in neurodegenerative disorders, such as Parkinson's 5,6 , Alzheimer's 7,8 , and Huntington's diseases or amyotrophic lateral sclerosis 9 . Activated microglia release inflammatory mediators steadily over time, which boosts the inflammatory cycle 10 . Unlike the protective role of acute neuroinflammation, chronic neuroinflammation mostly damages nervous tissues 11 . Also, retinal neurodegenerative diseases, such as retinitis pigmentosa (RP) 3 , age-related macular degeneration 12 , and glaucoma 13,14 are concomitant with chronic microglial activation and neuroinflammatory process. Retinal microglial cells may also be activated by systemic infection by fungus or virus 15,16 and by immunosuppression or peripheral inflammation 17 , which could worsen the degenerative process. However, the mechanisms by which systemic inflammation and microglial activation exert their effects on chronic neurodegeneration are not yet widely understood.
Lipopolysaccharide (LPS) administration is commonly used as a model of neuroinflammation [18][19][20] . LPS induces microglial activation by a Toll-like receptor 4 (TLR4)dependent pathway, and is accompanied by the production and release of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 and Tumor necrosis factor alpha (TNF-α), by the Mitogen-activated protein kinase (MAPK) and Nuclear factor kappa beta (NF-kβ) routes 21,22 . Systemic administration of LPS also activates retinal microglia 23,24 , but the exact effect of LPS-activated microglial cells on photoreceptor death and the mechanisms underlying microglia-photoreceptor crosstalk remain to be determined.
The main objective of this work was to elucidate whether a peripheral inflammatory process must be considered a risk factor in the progression of retinal degenerative diseases. We studied the consequences of systemic administration of LPS in P23H rats, an animal model of RP. We decided to administer low doses of LPS in order to trigger the alert status of microglia, but avoid a massive influx of pro-inflammatory cells to the retina, in an attempt to mimic a mild chronic peripheral condition. Our data suggest that peripheral inflammation exacerbates the inflammatory response in the retina and accelerates neurodegenerative events in the P23H rat model, causing greater loss of photoreceptor numbers and increased retinal dysfunction.

Animals
Homozygous P23H line 3 rats, kindly provided by Matthew LaVail (UCSF School of Medicine; www.ucsfeye.net/ mlavailRDratmodels.shtml), were used as a RP model. Sprague-Dawley rats (SD) were used as a healthy control group (Harlan Laboratories, Barcelona, Spain). Twelve SD rats and 12 P23H rats were housed in cages under controlled photoperiod (12-h light/12-h dark), humidity (55-60%), and temperature (23 ± 1°C) conditions. Both food and water were provided ad libitum. All procedures received prior approval from the ethics committee for animal care and use at the University of Alicante (UA-07/ 22/2013). The animals were treated according to current guidelines and regulations for the use of laboratory animals (NIH, ARVO and European Directive 2010/63/EU), in an effort to minimize their suffering and limit the number of animals used.

LPS administration
LPS was purchased from the Sigma-Aldrich laboratory in St. Louis, MO (USA). A stock solution of 400 µg/ml was prepared in saline and stored in aliquots of 1 ml at −20°C until administration. A total of six SD and six P23H rats received three weekly injections of LPS intraperitoneally (i.p.) (60 µg/kg in saline) (LPS-injected rats), whereas six SD and six P23H rats were used as control groups receiving saline injections at the same time points (vehicle-injected rats). Treatment began at P20 and continued until P60.

Electroretinographic (ERG) records
ERGs were performed under scotopic conditions at P60, after completion of the treatments. Scotopic flash-induced ERG responses to light stimuli produced by a Ganzfeld stimulator were recorded in both eyes. Light stimuli were administered for 10 ms at 11 increasing luminances, ranging from −5.2 to 0 log cd s/m 2 . Between 3 and 10 consecutive recordings were averaged for each light level. A 10-s interval was provided between flashes in the case of dim flashes (−5.2 to −1.4 log cd s/m 2 ), and up to 20 s for the brightest flashes (−0.8 to 0 log cd s/m 2 ).

Quantification of microglial markers
To analyze microglial cells in each group of animals, we examined vertical retinal sections. The quantity of cells expressing either or both markers (Iba1+/MHC-II−, Iba1 −/MHC-II+ and Iba1+/MHC-II+) was determined at ×63 magnification. The data represent counts for the total length of the section analyzed and were then extrapolated to 1 mm of retinal tissue (as obtained through the ImageJ software; National Institutes of Health, Bethesda, MD, USA). We analyzed at least two non-consecutive retinal sections of each animal belonging to each experimental group, including the optic nerve, and the counts are those for the total length of each retinal section.

Quantification of gliosis in Müller cells and astrocytes
To assess the response of Müller cells and astrocytes to LPS administration, we quantified gliosis in Müller cells and astrocytes using anti-glial fibrillary acidic protein (GFAP) antibodies. To do that, we analyzed four animals per experimental group, evaluating in each retinal section four equidistant regions in the temporal-nasal axis: two in the temporal area (temporal-peripheral and temporal-central) and two in the nasal area (nasal-peripheral and nasal-central). Gliosis in Müller cells and astrocytes were evaluated by measuring the fluorescence area related with GFAP immunostaining using the ImageJ software.

Quantification of photoreceptor rows, synaptic connectivity, and structural integrity of cone photoreceptors
To study retinal degeneration, we quantified photoreceptor rows, using the nuclear marker TO-PRO 3, in at least two non-consecutive retinal sections of each animal. Quantifications were performed in six equidistant regions in the temporal-nasal axis: three in the temporal area (temporal-peripheral, temporal-medial, and temporal-central) and three in the nasal area (nasalperipheral, nasal-medial, and nasal-central). An average value of photoreceptor rows was calculated for each animal.
We also evaluated synaptic connectivity in the outer and inner plexiform layers (OPL/IPL) of the retina by measuring the fluorescence area associated with Bassoon immunostaining using the ImageJ software. In all sections analyzed, this quantification was performed close to the optic nerve. The structural integrity of cone photoreceptors was evaluated by immunostaining with a polyclonal rabbit anti-cone arrestin antibody.

Flow cytometry
To identify microglial population and cell activation by flow cytometry, a retinal cell suspension was subjected to a triple labeling with a cocktail of antibodies: anti-CD11b conjugated to APC (allophycocyanin-Blue, clone M1/70; eBioscience), anti-MHC Class II PE conjugated (phycoerythrin-Red, clone M5/114.15.2; Miltenyi Biotec, Bergish Gladbach, Germany) and anti-CD45 FITC conjugated (clone 104.2, Miltenyi Biotec). Each rat retina was analyzed individually. Data were acquired on a LSRFortessa flow cytometer (BD Biosciences) and analyzed using FACSDiva software (BD Biosciences).

qRT-PCR arrays
To analyze the mRNA expression levels of genes associated with apoptosis and inflammation pathways, we used two commercial arrays from Qiagen® (Custom RT 2 PCR Apoptosis array 96 wells; Qiagen, Hilden, Germany) and Applied Biosystems (TaqMan® Inflammation Array 96 wells FAST plates; Applied Biosystems, Carlsbad, CA, USA). Total RNA from retinas of vehicle-and LPS-treated animals was obtained using the mirVana total RNA Isolation kit (Ambion Ltd., Cambridgeshire, UK) and treated with Turbo DNase (Ambion Ltd). Total RNA quantification was carried out using a Nanodrop-1000 device (Thermo Fisher Scientific, Waltham, MA, USA). Two hundred nanograms of total RNA from each sample were reverse transcribed into complementary DNA (cDNA) using the RT 2 first strand kit (Qiagen) and High Capacity RNA-to-cDNA Kit (Applied Biosystems). We quantified the mRNA expression levels of TNF-α, IL-1α, IL-1β, Caspase-1, Caspase-8 and CX3CL1, p53, Bcl-2, Bad, Bax, Hrk, and Apaf-1, GSK3β, Akt and mTOR. RNA18S, Actβ, Hprt1, and Rpl13α were used as house-keeping genes. RNA18S was used to normalize the analysis. Each run also included negative controls. A StepOnePlus® system (Applied Biosystems) was used to conduct PCR experiments. The data analysis was performed using the ΔΔCt based calculations using free array data analysis web portal software, provide by Qiagen®. Data obtained from Applied Biosystems arrays were analyzed using the ExpressionSuite software (www.thermofisher. com/es/es/home/technical-resources/software-downloads/ expressionsuite-software.html) with the same calculation method.
Statistical analysis SPSS 20.0 software (IBM, Armonk, NY, USA) was used to perform the statistical analysis and a Multivariate Analysis of Variance (MANOVA) was calculated to assess the effects of LPS administration. A Bonferroni's test was used to perform post-hoc pairwise comparisons when the significance level was found to be 0.05. All the analyzed categories exhibited normal distributions and homogeneity of variance. Statistical significance was established at P < 0.05. The data were subsequently plotted as the mean ± SEM.
Flow cytometry results were analyzed using Student's t-tests to compare values in different animal groups. Results were expressed as the mean ± standard deviation and were considered to be statistically significant at P < 0.05.

The effects of systemic LPS administration on ERG retinal responses
To assess the effect of low-dose systemic LPS in the functional activity of the retina of P23H and SD rats, ERG responses induced by scotopic flash were recorded in four animal groups (vehicle-injected SD, LPS-injected SD, vehicle-injected P23H, and LPS-injected P23H; n = 6 in each group). As shown in Fig. 1, chronic administration of LPS to P23H rats reduced the ERG response to light flashes. The maximum a-and b-wave amplitudes registered were 31% and 33% lower (respectively) in LPS-injected P23H rats than that observed in vehicle-injected dystrophic animals (57.7 ± 1.7 μV and 38.3 ± 1.2 μV for the awave, 342.2 ± 5.1 μV and 227.7 ± 4.5 μV for the b-wave; analysis of variance (ANOVA), Bonferroni's test, P < 0.001, for both cases; Fig. 1). The same doses of LPS did not affect the retinal response of SD rats.
The effects of systemic LPS administration on the number of photoreceptor rows, structural integrity of cone photoreceptors, and synaptic connectivity The analysis of retinal sections of each animal group stained with TO-PRO 3 revealed that the retinas of vehicle-injected P23H animals had an average of 7.7 ± 1.1 rows of photoreceptors, whereas vehicle-injected SD rats showed an average of 11.1 ± 0.8 photoreceptor rows Fig. 1 Effect of LPS on functional changes associated with retinal degeneration. a Representative scotopic full-field ERG waveforms from P60 SD and P23H rats injected with vehicle or LPS from P20 to P60. Units on the left show input flash intensities in log cd s/m 2 . b, c Luminance-response curves for mixed scotopic b-waves b and a-waves c from vehicle-injected SD rats (black dots), LPS-injected SD rats (white dots), vehicle-injected P23H rats (black squares), and LPS-injected P23H rats (white squares). Data are represented as mean ± SEM, n = 6. *P < 0.05, ***P < 0.001; ANOVA, Bonferroni's test (Fig. 2). The systemic administration of LPS did not induce a significant reduction of photoreceptor rows in SD rats, whereas the same LPS dose reduced by 45% the number of photoreceptor rows in P23H rats (4.2 ± 0.8 rows; ANOVA, Bonferroni´s test, P < 0.001). This reduction in the quantity of photoreceptor rows was evident in all retinal sections analyzed (data not shown).
We also evaluated the number, shape, and structural integrity of cone photoreceptors in the retinas of each experimental group, using antibodies against cone arrestin. In SD rats, the administration of LPS did not provoke significant differences in morphology or number of cone photoreceptors (Figs. 3a, b). In vehicle-injected P23H rats, cones showed inner and outer segments than were shorter than those observed in SD rats (Figs. 3a, c). But cone photoreceptors experienced more drastic changes in LPStreated P23H rats. In these animals, the cones were very small, with short and swollen outer segments (Fig. 3d). The axons were missing entirely, and the pedicles emerged directly from the cone cell bodies. The number of cone photoreceptors was also smaller in LPS-treated P23H rats than in vehicle-treated P23H rats (60 ± 2.5 cones/mm and 109 ± 2 cones/mm, respectively; ANOVA, Bonferroni's test, P < 0.001, n = 6 in both cases; Fig. 3e). Given the significant decrease in the number of photoreceptor rows in P23H rats administered LPS, synaptic connections were assessed in the retina of dystrophic and normal animals. The analysis of retinal sections immunostained with polyclonal rabbit anti-calbindin antibodies showed no significant differences in the average number of horizontal cells between P23H or SD rats administered LPS or vehicle (Supplementary material 1). However, in LPS-injected P23H rats the dendritic arborization of horizontal cells was clearly degenerated as compared with vehicle-injected P23H rats (Fig. 4). Numerous Bassoon-immunopositive spots were observed at the INL level in P23H animals, regardless of whether they were administered LPS. By contrast, few synaptic ribbons were observed at the OPL in P23H rats injected with LPS ( Fig. 4d), thus revealing that presynaptic contacts between photoreceptors and bipolar/horizontal cells were reduced in these animals.
To quantify the differential expression of Bassoon, we obtained the profile plots of mean gray intensity in each horizontal line of images of the retina showing Bassoon immunofluorescence (Figs. 5a, b). As it is shown in Fig. 5c, the mean area of the profiles obtained at the OPL level in vehicle-injected P23H rats were significantly higher (3.5fold greater) than those obtained in P23H rats administered LPS (11200 ± 500 pixels and 3500 ± 150 pixels, respectively; ANOVA, Bonferroni's test, P < 0.001, n = 6 in both cases). No significant differences were observed at the IPL (Fig. 5d, 15500 ± 700 pixels and 14500 ± 850 pixels, respectively). In the SD rat groups, we did not find any significant differences in the fluorescent area of either plexiform layer (data not shown).

The effects of systemic LPS administration on retinal microglial number and activation state
In SD rat retinas, Iba1+ cells were distributed in the inner and outer plexiform and ganglion cell layers (Fig. 6a) and showed negligible immunoreactivity against MHC Class II antibodies (Figs. 6b, c). In these animals, LPS injection did not induce any differences in the morphology, activation state, density, or distribution of retinal microglial cells (Figs. 6d-f). In P23H retinas, microglial cells were seen in all retinal layers, and were present in greater numbers in IPL and OPL (Figs. 6g-i), in a density higher than that observed in SD rats (11.8 ± 1 Iba1+ cells/ mm and 7.7 ± 0.5 Iba1+ cells/mm, respectively; ANOVA, Bonferroni's test, P < 0.01, n = 6, in both cases; Fig. 7a). Moreover, P23H rat retinas presented ameboid Iba1+ cells, some of them exhibiting the activation marker MHC-II (Figs. 6g-i). LPS injection in P23H rats increased the quantity of Iba1+ cells, many of them showing ameboid shape and expressing MHC-II (Figs. 6j-l). Quantitative analysis of the retinal sections showed that in LPSinjected P23H rats the relative quantity of Iba1+/MHC-II − cells was 12% higher than that observed in vehicleinjected P23H rats (ANOVA, Bonferroni's test, P < 0.05; Fig. 7b). The number of double immunoreactive cells Iba1 +/MHC-II+ was also significantly higher in LPS-injected P23H animals when compared with P23H rats (35% more; ANOVA, Bonferroni´s test, P < 0.001; Fig. 7b).
In addition to the morphological and quantitative analysis of retinal sections, retinal cells expressing the microglia marker CD11b were identified by flow cytometry (Figs. 8a, b) and MHC-II and CD45 expression was measured to determine their level of activation (Figs. 8a,  c). SD rats showed no significant differences in the number of retinal CD11b+ cells between LPS-and vehicle-injected animals (0.8 ± 0.2% in vehicle-injected vs. 0.9 ± 0.2% in LPS-injected animals, Fig. 8b). Between these groups, the amount and distribution of different phenotypes of microglial cells also did not show any differences. Otherwise, P23H retinas showed an increased CD11b+ microglial population (4.0 ± 1.2%) with respect to SD rat retinas (Student's t-test, P < 0.001; Fig. 8b). The quantity of CD11b+ cells was not significantly different between P23H rats injected with vehicle or LPS, but the fluorescence intensity for CD11b and CD45 was significantly higher in LPS-injected vs. vehicle-injected P23H rats (Student's t-test, P < 0.01 and P < 0.001, respectively; Fig. 8c).

The effects of systemic LPS administration on astrocytes and Müller cells
The expression of GFAP in healthy rat retinas is restricted to astrocytes within the inner retina 3 . However, under many pathological conditions or retinal damage, Müller cells are found to express GFAP 3,27 . Using anti-GFAP antibodies, we assessed the response of Müller cells and astrocytes to LPS treatment (Fig. 9). In vehicleinjected SD rats, GFAP immunoreactivity was virtually limited to the inner margin of the retina, colocalizing with astrocytes, whereas P23H rat retinas also showed GFAP immunoreactivity in Muller cells, which exhibited a marked labeling throughout the entire cell. In both strains, SD and P23H rats, LPS injection triggered an increase in GFAP immunoreactivity in the Müller cell processes as compared with vehicle-injected animals (37652 ± 2000 pixels and 18296 ± 3100 pixels, respectively, in SD animals and 71655 ± 1500 pixels and 55185 ± 1220 pixels, respectively, in P23H animals; ANOVA, Bonferroni's test, P < 0.001, n = 4 in both cases; Figs. 9a-e). LPS treatment also triggered an increase in GFAP immunofluorescence in the inner margin of the retina, colocalizing with astrocytes and end feet of Müller cells (68834 ± 1900 pixels and 29613 ± 1500 pixels, respectively, in SD animals and 86308 ± 4500 pixels and  62251 ± 2600 pixels, respectively, in P23H animals; ANOVA, Bonferroni's test, P < 0.001, n = 4 in both cases; Fig. 9f).

Discussion
The potential influence of systemic pathologies in the progression of retinal neurodegenerative diseases is a crucial issue to be explored due to its expected influence on the prognosis and therapy. Our main objective was to assess whether peripheral inflammatory conditions can worsen the progression of neurodegeneration in an animal model of RP. In order to mimic the effect of a mild chronic condition, we chronically administered low doses of LPS, which did not induce physiological or morphological changes in healthy retinas.

LPS-induced systemic inflammation aggravates morphological and physiological disturbances in dystrophic retinas
Compared with healthy rat retinas, P23H transgenic rat retinas showed a decreased number of photoreceptors and synaptic contacts, as well as lower ERG responses. Hereditary retinal degeneration was accompanied by microglial activation, evidenced by increased microglial density and higher expression levels of microglial activation markers. This is in agreement with previous findings showing that neurodegenerative retinal diseases are linked to chronic  3,4,28,29 . Activated microglia mediate neuroinflammation by releasing several pro-inflammatory molecules including cytokines, chemokines, trophic factors, and small molecules, which promote and perpetuate the inflammatory response, potentially leading to neurodegeneration 3,10,30-32 .
As a result of repeatedly administering low doses of LPS to P23H degenerating retinas, a further dramatic decrease in the quantity of photoreceptor cells and synaptic connectivity in the OPL was observed, in agreement with the lower ERG responses observed in this experimental group. Specifically, it has been demonstrated that systemic inflammation induced by LPS increases neuronal apoptosis in the brain of an animal model of prion disease 33 . Hippocampal-dependent learning deficits are triggered by LPS-induced peripheral inflammation in elderly rats 34 , and repeated challenges with LPS cause increased Microtubule associated protein (MAP, Tau) hyperphosphorylation in a transgenic model of Alzheimer's disease 35 . SD control rats administered with the same doses of LPS failed to show retinal changes at either a structural or physiological level.

LPS-induced systemic inflammation triggers microglial activation and gliosis in dystrophic retinas
The LPS-induced microglial activation observed in P23H rat retinas is in agreement with our previous works and those by others, showing that systemic infections caused by cytomegalovirus 15 or Candida albicans 16 , as well as peripheral insults 17 are capable of triggering microglial activation and increasing the quantity of microglial cells in the retina. The higher number of Iba1positive cells in the retina of LPS-injected P23H rats could be caused by migration of inflammatory macrophages, b Mean values of CD11bimmunopositive population in vehicle-injected and LPS-injected SD and P23H rats. c Histograms representing mean fluorescence intensity values of the CD11b-positive population labeled with anti-CD45, anti-MHC-II, and anti-CD45 antibodies (10 6 cells were analyzed in each assay) in both P23H experimental groups. Data are presented as mean values ± SEM. **P < 0.01, ***P < 0.001; Student's t-test myeloid precursors, or inflammatory monocytes, or by the proliferation of retinal microglia, as these cell types are able to proliferate in situ once they have differentiated 36 . In a previous work, we evidenced the presence of proliferative Iba1+ cells in P23H rat retinas, but not in healthy control rats, suggesting that increases of Iba1positive cells in P23H rats is accomplished, at least partially, by the in situ proliferation of resident microglia 4 .
In this study, we show that LPS injection induces an increase in GFAP immunoreactivity in the Müller cell processes in the inner margin of the retina of P23H rats. A hallmark of gliosis is the upregulation of intermediate filament proteins in glial cells, including GFAP. Thus, our results suggest that low-dose LPS treatment exacerbates reactive gliosis in astrocytes and Müller cells.

Systemic inflammation exacerbates inflammatory and apoptotic pathways in dystrophic retinas
At the molecular level, low-dose LPS treatment in SD rats induced upregulation of several important apoptosisrelated genes, involved in both death and survival signals (Bcl-2, Bax, Hrk, p53, Apaf-1, and Caspase-8), which can be associated with an early cellular response. In this experimental group, we could not detect any increased expression of inflammation-related genes. This result is consistent with the normal appearance of these retinas, and probably due to the low doses of LPS we used. As we did not detect any functional nor morphological change in the retinas, we assume that retinal cells were able to maintain cellular homeostasis. P23H rat retinas showed increased expression levels of several inflammation-related genes (Tnf-α, Il-1α, Il-1β, Caspase-1, and Caspase-8) in agreement with the higher inflammatory state of the degenerating retina that is observed during all the degenerative process, compared with healthy retinas 4 . This result also agrees with the high levels of proinflammatory cytokines present in certain neurodegenerative diseases, such as Parkinson's 6,37 , Alzheimer's 7,8 , and Huntington's diseases or amyotrophic lateral sclerosis 9 . In P23H rat retinas, we also detected increased levels of expression of genes associated with apoptosis (p53, Bcl-2, Bad, Bax, and Apaf-1), in concordance with the enhanced rate of apoptosis observed in this work and in previous published studies 38 . In P23H rats, LPS treatment induced a drastic further increase in the expression of inflammation-and apoptosis-related genes, which agrees and their effects on autophagy, necroptosis, inflammation, and apoptosis as described by others (see text for references). a-c Relative expression (fold change, in red squares) of mRNA in the retina of LPS-injected SD rats a, LPS-injected P23H rats b and vehicle-injected P23H rats c, compared with the expression levels found in vehicle-administered SD rats. d Normalized expression of mRNA in LPS-injected P23H rats, compared with that found in vehicle-administered P23H rats. The relative size of the letters represents the relative level of gene expression with other authors' results showing that systemic administration of LPS, even in a single dose, causes increased microglial activation, inflammatory exacerbation, and enhanced neurodegeneration through increased production of TNF-α, IL-1β, and IL-6 in the brain 39,40 . In P23H model of retinal degeneration, an increased expression of Tnf-α, Il-1α, and Il-1β could, at least in part, explain the increased loss of photoreceptors observed in LPS-injected P23H rats 3,10,[30][31][32][41][42][43] . In LPS-injected P23H rats, we found further upregulation of the Caspase-1 gene, pointing to a scenario of even greater apoptosis and inflammation [44][45][46] .
In agreement with the accepted main role of p53 in neuroinflammatory processes in neurodegeneration 47 , in our experimental conditions, the transcription factor p53 also appears as a key factor in the neurodegenerative process. p53 expression was enhanced in all the conditions tested, with higher increases in P23H rat retinas than in LPS-injected SD rat retinas, and even higher expression levels in the retinas of LPS-injected P23H rats. Hence, in our experiments, the response obtained with repeated low doses of LPS reinforce the idea that a chronic inflammatory peripheral state does affect the degenerative process by increasing the expression of inflammation-and apoptosis-related markers.
The activation of TLR4 by LPS can participate in cell apoptosis through the phosphatidylinositol 3 kinase/ AKT/GSK3β signaling pathway 52 . In our experiments, LPS administration in low doses to SD rats provoked an imbalance between pro-death and pro-survival signals can be determinant for the final outcome of the cell. Additionally, the Akt, Glycogen Synthase Kinase 3 beta (GSK3β), and Mammalian Target of Rapamycin (mTOR) signaling pathways have been implicated in the final outcome of threatened cells [52][53][54][55][56][57][58][59] . Inhibitors of Glycogen Synthase Kinase 3 beta (GSK3β) provide potential therapeutic strategies to control inflammation 60 , and are thus drugs that may potentially be used for the treatment of RP 61 . Fig. 11 Schematic representation of the hypothetical contribution of death pathways to the outcome of the cell in different noxious situations in animal retinas: a chronic administration of low doses of LPS (from P20 to P60) to SD healthy rats, b genetically determined retinal dystrophy (P23H rat model of RP), and c chronic administration of low doses of LPS to the genetically determined dystrophic rats (P23H model). Our results indicate that in healthy retinas, low doses of LPS induce expression changes in pro-and anti-death signals, as well as in apoptosis-related transcripts, although no signs of cell death are observed. In P23H rat retinas, increased expression levels of genes related to apoptosis and inflammation and pro-death signals alter the balance between pro-survival and pro-death signals and influence the death scenario observed at P60. In this model of degeneration, the administration of low doses of LPS probably induces an increase in necroptotic cell death, together with insufficient autophagic mechanisms that would worsen the retinal degenerative process and contribute to the exacerbation of the morphological and functional damage, concordant with the morphological worsening and loss of function observed Our results revealed that LPS administration induced increased expression levels of mTOR in the retina of SD rats, in agreement with other works that attribute to mTOR a main role in the inflammatory effects of LPS 58,62,63 . The expression levels of mTOR were higher in both P23H rat groups, which could contribute to the increased degeneration observed in these animals, as previously described for retinal degenerative processes 59 . We hypothesize that the effects of LPS may alter the balance between pro-survival and pro-death signals and exacerbate the cell death processes (Fig. 11).
Systemic LPS can modify death pathways contribution to the degenerative process in dystrophic retinas In this death scenario, the contribution of other death pathways as necroptosis and autophagy should be taken into account. When compared with vehicle-injected P23H rat retinas, the retinas of P23H rats injected with LPS showed an enhanced death stimulus, with dramatically increased expression levels of Tnf-α and p53 51,64 . As both apoptosis and necroptosis can be triggered simultaneously in the retina 65,66 , these data, together with the downregulation of Caspase-8, and the reduced expression of the pro-apoptotic Apaf-1 in these animals, leads us to hypothesize that LPS trigger the activation of necroptosis pathways in the retina of dystrophic rats. Necroptosis cell death would contribute to the augmented inflammatory response in the retina of these animals and to the worsening of the morphological and functional activity. This hypothesis agrees with prior studies showing that retinal microglia undergo RIP1/3-mediated necroptosis in rd1 mouse model of retinal neurodegeneration and also after an acute retinal neural injury 67 . Necroptotic microglia release large amounts of pro-inflammatory cytokines, such as TNF-α, in response to LPS or oxidative stress, triggering neuroinflammation and, consequently, exacerbating retinal damage and disease progression 67 .
Previous studies showed that LPS treatment can promote autophagy in microglia and macrophages 68,69 . In our experiments, LPS administration to P23H dystrophic rats induced increased levels of Beclin-1 and Bad expression and reduced expression levels of mTOR. These results suggest a higher degree of autophagy processes 70 . But the higher levels of Bax, Bcl-2, and p53 31,70-76 seem to point to the fact that, despite the increased pro-autophagy markers apoptotic and inflammatory state in these animals is dampening the activation of appropriate autophagy mechanisms. Autophagy dysfunction has been related to a number of neurodegenerative diseases, such as Alzheimer's or Parkinson's diseases, among other neuron-affecting pathologies 7,9,69,77,78 .
In the degenerating retinas of P23H rats treated with LPS, the insufficient autophagy together with the promotion of necroptotic cell death would worsen the degenerative process and contribute to the exacerbation of the morphological and functional damage.

Conclusions
All these data indicate that administering low doses of LPS to dystrophic P23H rats exacerbates inflammatory and apoptotic states, accelerates the loss of cone photoreceptors and retinal functionality, and triggers the worsening of negative symptoms of the disease. Given that LPS administration emulates the noxious influence of a peripheral inflammatory condition, these results are particularly important for patients that suffer from ocular neurodegenerative diseases such as glaucoma, diabetic retinopathy, RP, or macular degeneration. According to these results, peripheral damage, such as a systemic infection or chronic inflammatory process, could accelerate the progression of the disease and should be considered when selecting an appropriate therapy to revert, block or slow-down the degenerative process.
Neurodegenerative diseases share common pathogenic mechanisms, including neuroinflammation. In this sense, the activation of microglial cells has been extensively reported in both retinal and brain diseases 3,79 . On the other hand, it has been previously shown that systemic conditions can worsen and exacerbate the symptoms of chronic neurodegeneration in degenerative diseases as Parkinson's and Alzheimer's disease, accelerating the progression of the disease 40,80-83 . Therefore, our results suggest a possible harmful effect of systemic inflammation on the progression of retinal degenerative diseases that could be extrapolated to brain neurodegenerative disorders, such as Parkinson's or Alzheimer's diseases.