Serum Vitamin D, Folate and Fatty Acid Levels in Children with Autism Spectrum Disorders: A Systematic Review and Meta-Analysis.

Vitamin and fatty acid deficiency in children diagnosed with autism has been linked to the etiology and course of the disease but the results have been inconsistent. In our work, we present a narrative review, which includes 20 observational studies that provide data on the blood levels of vitamin D, folate, or fatty acids of children diagnosed with ASD (Autism Spectrum Disorder-AG group), and of a control group (children without this disorder-CG group). The main characteristics and results are presented in a summary table. Of the 20 above-mentioned studies, a meta-analysis of vitamin D and folate levels was carried out in 14 of them, with a total of 2269 children (AG = 1159, CG = 1110). Vitamin D levels were lower in AG compared to CG: SMD, 95% CI = - 0.83 [- 1.15, - 0.50]. In terms of folate levels, a total of 299 children (AG = 148, CG = 151) were analyzed, finding no significant differences with the control group: SMD, 95% CI = - 0.16 [- 0.63, 0.32]. Only one study that provided data on fatty acids in children with ASD was included in the review although it was not possible to include it in the meta-analysis. We conclude that the nutritional status (vitamin and fatty acid levels) of patients diagnosed with ASD should be taken into account, as correct adjustment of these levels-may produce an improvement in the course of the disease and could also reduce the risk of its development.

Poly(ADP-Ribose) Polymerase-1 inhibition potentiates cell death and phosphorylation of DNA damage response proteins in oxidative stressed retinal cells.

Oxidative stress (OxS) is involved in the development of cell injures occurring in retinal diseases while Poly(ADP-ribose) Polymerase-1 (PARP-1) is a key protein involved in the repair of the DNA damage caused by OxS. Inhibition of PARP-1 activity with the pharmacological inhibitor PJ34 in mouse retinal explants subjected to H2O2-induced oxidative damage resulted in an increase of apoptotic cells. Reduction of cell growth was also observed in the mouse cone like cell line 661 W in the presence of PJ34 under OxS conditions. Mass spectrometry-based phosphoproteomics analysis performed in 661 W cells determined that OxS induced significant changes in the phosphorylation in 1807 of the 8131 peptides initially detected. Blockade of PARP-1 activity after the oxidative treatment additionally increased the phosphorylation of multiple proteins, many of them at SQ motifs and related to the DNA-damage response (DDR). These motifs are substrates of the kinases ATM/ATR, which play a central role in DDR. Western blot analysis confirmed that the ATM/ATR activity measured and the phosphorylation at SQ motifs of ATM/ATR substrates was augmented when PARP-1 activity was inhibited under OxS conditions, in 661 W cells. Phosphorylation of ATM/ATR substrates, including the phosphorylation of the histone H2AX were also induced in organotypic cultures of retinal explants subjected to PARP-1 inhibition during exposure to OxS. In conclusion, inhibition of PARP-1 increased the phosphorylation and hence the activation of several proteins involved in the response to DNA damage, like the ATM protein kinase. This finally resulted in an augmented injury in mouse retinal cells suffering from OxS. Therefore, the inhibition of PARP-1 activity may have a negative outcome in the treatment of retinal diseases in which OxS is involved.

Poly(ADP-ribose)polymerases inhibitors prevent early mitochondrial fragmentation and hepatocyte cell death induced by H2O2.

Poly(ADP-ribose)polymerases (PARPs) are a family of NAD+ consuming enzymes that play a crucial role in many cellular processes, most clearly in maintaining genome integrity. Here, we present an extensive analysis of the alteration of mitochondrial morphology and the relationship to PARPs activity after oxidative stress using an in vitro model of human hepatic cells. The following outcomes were observed: reactive oxygen species (ROS) induced by oxidative treatment quickly stimulated PARPs activation, promoted changes in mitochondrial morphology associated with early mitochondrial fragmentation and energy dysfunction and finally triggered apoptotic cell death. Pharmacological treatment with specific PARP-1 (the major NAD+ consuming poly(ADP-ribose)polymerases) and PARP-1/PARP-2 inhibitors after the oxidant insult recovered normal mitochondrial morphology and, hence, increased the viability of human hepatic cells. As the PARP-1 and PARP-1/PARP-2 inhibitors achieved similar outcomes, we conclude that most of the PARPs effects were due to PARP-1 activation. NAD+ supplementation had similar effects to those of the PARPs inhibitors. Therefore, PARPs activation and the subsequent NAD+ depletion are crucial events in decreased cell survival (and increased apoptosis) in hepatic cells subjected to oxidative stress. These results suggest that the alterations in mitochondrial morphology and function seem to be related to NAD+ depletion, and show for the first time that PARPs inhibition abrogates mitochondrial fragmentation. In conclusion, the inhibition of PARPs may be a valuable therapeutic approach for treating liver diseases, by reducing the cell death associated with oxidative stress.

Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP.

Microglial cell precursors located in the area of the base of the pecten and the optic nerve head (BP/ONH) start to enter the retina of quail embryos at the 7th day of incubation (E7), subsequently colonizing the entire retina by central-to-peripheral tangential migration, as previously shown by our group. The present study demonstrates a precise chronological coincidence of the onset of microglial cell entry into the retina with a striking increase in death of retinal cells, as revealed by their active caspase-3 expression and TUNEL staining, in regions dorsal to the BP/ONH area, suggesting that dying retinal cells would contribute to the microglial cell inflow into the retina. However, the molecular mechanisms involved in this inflow are currently unclear. Extracellular nucleotides, such as ATP and UDP, have previously been shown to favor migration of microglia towards brain injuries because they are released by apoptotic cells and stimulate both chemotaxis and chemokinesis in microglial cells via signaling through purinergic receptors. Hence, we tested here the hypothesis that ATP and UDP play a role in the entry and migration of microglial precursors into the developing retina. For this purpose, we used an experimental model system based on organotypic cultures of E6.5 quail embryo retina explants, which mimics the entry and migration of microglial precursors in the in situ developing retina. Inhibition of purinergic signaling by treating retina explants with either apyrase, a nucleotide-hydrolyzing enzyme, or suramin, a broad spectrum antagonist of purinergic receptors, significantly prevents the entry of microglial cells into the retina. In addition, treatment of retina explants with either exogenous ATP or UDP results in significantly increased numbers of microglial cells entering the retina. In light of these findings, we conclude that purinergic signaling by extracellular ATP and UDP is necessary for the entry and migration of microglial cells into the embryonic retina by inducing chemokinesis in these cells.

Microglial Activation Promotes Cell Survival in Organotypic Cultures of Postnatal Mouse Retinal Explants.

The role of microglia during neurodegeneration remains controversial. We investigated whether microglial cells have a neurotoxic or neuroprotective function in the retina. Retinal explants from 10-day-old mice were treated in vitro with minocycline to inhibit microglial activation, with LPS to increase microglial activation, or with liposomes loaded with clodronate (Lip-Clo) to deplete microglial cells. Flow cytometry was used to assess the viability of retinal cells in the explants and the TUNEL method to show the distribution of dead cells. The immunophenotypic and morphological features of microglia and their distribution were analyzed with flow cytometry and immunocytochemistry. Treatment of retinal explants with minocycline reduced microglial activation and simultaneously significantly decreased cell viability and increased the presence of TUNEL-labeled cell profiles. This treatment also prevented the migration of microglial cells towards the outer nuclear layer, where cell death was most abundant. The LPS treatment increased microglial activation but had no effect on cell viability or microglial distribution. Finally, partial microglial removal with Lip-Clo diminished the cell viability in the retinal explants, showing a similar effect to that of minocycline. Hence, cell viability is diminished in retinal explants cultured in vitro when microglial cells are removed or their activation is inhibited, indicating a neurotrophic role for microglia in this system.

DNA damage, poly(ADP-Ribose) polymerase activation, and phosphorylated histone H2AX expression during postnatal retina development in C57BL/6 mouse.

PURPOSE: The purpose of this study was to investigate the incidence of DNA damage during postnatal development of the retina and the relationship between DNA damage and cell death. METHODS: DNA damage in the developing postnatal retina of C57BL/6 mice was assessed by determining the amounts of 8-hydroxy-2'-deoxyguanosine (8-OHdG), which is indicative of DNA oxidation and related to the formation of DNA single-strand breaks (SSBs), and phosphorylated histone H2AX (γ-H2AX), a marker of DNA double-strand breaks (DSBs). Poly(ADP-ribose) polymerase (PARP) activation was measured by ELISA and Western blotting. The location of γ-H2AX-positive and dying cells was determined by immunofluorescence and TUNEL assays. RESULTS: Oxidative DNA damage was maintained at low levels during high PARP activation between postnatal days 0 (P0) and P7. Phosphorylated histone H2AX gradually increased between P0 and P14 and decreased thereafter. Phosphorylated histone H2AX-positive cells with cell death morphology or TUNEL positivity were more abundant at P7 than at P14. CONCLUSIONS: Oxidative DNA damage in postnatal retina increases during development. It is low during the first postnatal week when PARP-1 activity is high but increases thereafter. The rise in DSBs when PARP activity is downregulated may be attributable to accumulated oxidative damage and SSBs. At P7 and P14, γ-H2AX-positive cells are repairing naturally occurring DNA damage, but some are dying (mostly at P7), probably due to an accumulation of irreparable DNA damage.

Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.

Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.

Microglial cells in organotypic cultures of developing and adult mouse retina and their relationship with cell death.

Organotypic cultures of retinal explants allow the detailed analysis of microglial cells in a cellular microenvironment similar to that in the in situ retina, with the advantage of easy experimental manipulation. However, the in vitro culture causes changes in the retinal cytoarchitecture and induces a microglial response that may influence the results of these manipulations. The purpose of this study was to analyze the influence of the retinal age on changes in retinal cytoarchitecture, cell viability and death, and microglial phenotype and distribution throughout the in vitro culture of developing and adult retina explants. Explants from developing (3 and 10 postnatal days, P3 and P10) and adult (P60) mouse retinas were cultured for up to 10 days in vitro (div). Dead or dying cells were recognized by TUNEL staining, cell viability was determined by flow cytometry, and the numbers and distribution patterns of microglial cells were studied by flow cytometry and immunocytochemistry, respectively. The retinal cytoarchitecture was better preserved at prolonged culture times (10 div) in P10 retina explants than in P3 or adult explants. Particular patterns of cell viability and death were observed at each age: in general, explants from developing retinas showed higher cell viability and lower density of TUNEL-positive profiles versus adult retinas. The proportion of microglial cells relative to the whole population of retinal cells was higher in explants fixed immediately after their dissection (i.e., non-cultured) from adult retinas than in those from developing retinas. This proportion was always higher in non-cultured explants than in explants at 10 div, suggesting the death of some microglial cells during the culture. Activation of microglial cells, as revealed by their phenotypical appearance, was observed in both developing and adult retina explants from the beginning of the culture. Immunofluorescence with the anti-CD68 antibody showed that some activated microglial cells were CD68-positive but others were CD68-negative. Flow cytometry using CD68-labeling revealed that the percentage of CD68-positive microglial cells was much higher in developing than in adult retina explants, despite the activation of microglia in both types of explants, indicating that CD68-labeling was more closely related to the maturity degree of microglia than to their activation. Some swollen activated microglial cells entered the outer nuclear layer in developing and adult cultured retinal explants, whereas this layer was devoid of microglia in non-cultured explants. There was no apparent correlation between the distribution of microglia and that of TUNEL-labeled profiles. However, some swollen activated microglial cells in the outer and inner nuclear layers engulfed clusters of cell nuclei that were negative or weakly positive for TUNEL. This engulfment activity of microglia mimicked that observed in degenerative pathologies of the retina. We conclude that organotypic cultures from developing retinas show a higher rate of cell viability and better preservation of the normal cytoarchitecture in comparison to those obtained from adult retinas. In addition, the features of microglial response in cultured retinal explants show them to be a useful model for studying interactions between microglial cells and degenerating neurons in retinal diseases.

Migration and ramification of microglia in quail embryo retina organotypic cultures.

Organotypic cultures of retina explants preserve the complex cellular microenvironment of the retina and have been used as a tool to assess the biological functions of some cell types. However, studies to date have shown that microglial cells activate quickly in response to the retina explantation. In this study, microglial cells migrated and ramified in quail embryo retina organotypic cultures (QEROCs) according to chronological patterns bearing a resemblance to those in the retina in situ, despite some differences in cell density and ramification degree. Retinal explants from quail embryos at 9 days of incubation (E9) proved to be the best in vitro system for reproducing a physiological-like behavior of microglial cells when cultured in Eagle's basal medium supplemented with horse serum. During the first week in vitro, microglial cells migrated tangentially in the vitreal part of QEROCs, and some began to migrate radially from 3 days in vitro (div) onward, ramifying in the inner and outer plexiform layers, thus mimicking microglia development in the retina in situ, although reaching a lower degree of ramification after 7 div. From 8 div onward, microglial cells rounded throughout the explant thickness simultaneously with the nonphysiological appearance of dead photoreceptors and round microglia in the outernuclear layer. Therefore, E9 QEROCs can be used during the first week in vitro as a model system for experimental studies of molecules putatively involved in microglial migration and ramification.

Microglial response to light-induced photoreceptor degeneration in the mouse retina.

The microglial response elicited by degeneration of retinal photoreceptor cells was characterized in BALB/c mice exposed to bright light for 7 hours and then kept in complete darkness for survival times ranging from 0 hours to 10 days. Photodegeneration resulted in extensive cell death in the retina, mainly in the outer nuclear layer (ONL), where the photoreceptor nuclei are located. Specific immunolabeling of microglial cells with anti-CD11b, anti-CD45, anti-F4/80, anti-SRA, and anti-CD68 antibodies revealed that microglial cells were activated in light-exposed retinas. They migrated to the ONL, changed their morphology, becoming rounded cells with short and thick processes, and, finally, showed immunophenotypic changes. Specifically, retinal microglia began to strongly express antigens recognized by anti-CD11b, anti-CD45, and anti-F4/80, coincident with cell degeneration. In contrast, upregulation of the antigen recognized by anti-SRA was not detected by immunocytochemistry until 6 hours after light exposure. Differences were also observed at 10 days after light exposure: CD11b, CD45, and F4/80 continued to be strongly expressed in retinal microglia, whereas the expression of CD68 and SRA had decreased to near-normal values. Therefore, microglia did not return to their original state after photodegeneration and continued to show a degree of activation. The accumulation of activated microglial cells in affected regions simultaneously with photoreceptor degeneration suggests that they play some role in photodegeneration.

Embryonic and postnatal development of microglial cells in the mouse retina.

Macrophage/microglial cells in the mouse retina during embryonic and postnatal development were studied by immunocytochemistry with Iba1, F4/80, anti-CD45, and anti-CD68 antibodies and by tomato lectin histochemistry. These cells were already present in the retina of embryos aged 11.5 days (E11.5) in association with cell death. At E12.5 some macrophage/microglial cells also appeared in peripheral regions of the retina with no apparent relationship with cell death. Immediately before birth microglial cells were present in the neuroblastic, inner plexiform (IPL), and ganglion cell (GCL) layers, and their distribution suggested that they entered the retina from the ciliary margin and the vitreous. The density of retinal microglial cells strongly decreased at birth, increased during the first postnatal week as a consequence of the entry of microglial precursors into the retina from the vitreous, and subsequently decreased owing to the cessation of microglial entry and the increase in retina size. The mature topographical distribution pattern of microglia emerged during postnatal development of the retina, apparently by radial migration of microglial cells from the vitreal surface in a vitreal-to-scleral direction. Whereas microglial cells were only seen in the GCL and IPL at birth, they progressively appeared in more scleral layers at increasing postnatal ages. Thus, microglial cells were present within all layers of the retina except the outer nuclear layer at the beginning of the second postnatal week. Once microglial cells reached their definitive location, they progressively ramified.

Behavior of in vitro cultured ameboid microglial cells migrating on Müller cell end-feet in the quail embryo retina.

Ameboid microglial cells migrate tangentially on the vitreal part of quail embryo retinas by crawling on Müller cell end-feet (MCEF) to which they adhere. These microglial cells can be cultured immediately after dissection of the eye and isolation of sheets containing the inner limiting membrane (ILM) covered by a carpet of MCEF (ILM/MCEF sheets), to which the cells remain adhered. Morphological changes of microglial cells cultured on ILM/MCEF sheets for 4 days were characterized in this study. During the first minutes in vitro, lamellipodia-bearing bipolar microglial cells became rounded in shape. From 1 to 24 h in vitro (hiv), microglial cells swept and phagocytosed the MCEF on which they were initially adhered, becoming directly adhered on the ILM. MCEF sweep was dependent on active cell motility, as shown by inhibition of sweep after cytochalasin D treatment. From 24 hiv on, after MCEF phagocytosis, microglial cells became more flattened, increasing the surface area of their adhesion to substrate, and expressed the beta1 subunit of integrins on their membrane. Morphological evidence suggested that microglial cells migrated for short distances on ILM/MCEF sheets, leaving tracks produced by their strong adhesion to the substrate. The simplicity of the isolation method, the immediate availability of cultured microglial cells, and the presence of multiple functional processes (phagocytosis, migration, upregulation of surface molecules, etc.) make cultures of microglial cells on ILM/MCEF sheets a valuable model system for in vitro experimental investigation of microglial cell functions.