Round Microglia Research Articles

Melatonin Activates Anti-Inflammatory Features in Microglia in a Multicellular Context: Evidence from Organotypic Brain Slices and HMC3 Cells.

Melatonin (MEL) is a neurohormone endowed with neuroprotective activity, exerted both directly on neuronal cells and indirectly through modulation of responsive glial cells. In particular, MEL's effects on microglia are receptor-mediated and in part dependent on SIRT1 activation. In the present study, we exploited the highly preserved cytoarchitecture of organotypic brain cultures (OC) to explore the effects of MEL on hippocampal microglia in a 3D context as compared to a single cell type context represented by the human HMC3 cell line. We first evaluated the expression of MEL receptor MT1 and SIRT1 and then investigated MEL action against an inflammatory stimulation with LPS: OCs were cultured for a total of 2 weeks and during this time exposed to 0.1 μg/mL of LPS for 24 h either on day 1 (LPS 1°) or on day 11 (LPS 11°). MEL was added immediately after plating and kept for the entire experiment. Under these conditions, both MEL and LPS induced amoeboid microglia. However, the same round phenotype matched different polarization features. LPS increased the number of nuclear-NF-kB+ round cells and MEL alone or in combination with LPS increased BDNF+ round microglia. In addition, MEL contrasted LPS effects on NF-kB expression. Data from HMC3 microglia confirmed MEL's anti-inflammatory effects against LPS in terms of CASP1 induction and BDNF release, identifying SIRT1 as a mediator. However, no effects were evident for MEL alone on HMC3 microglia. Overall, our results point to the importance of the multicellular context for full MEL activity, especially in a preventive view, and support the use of OCs as a favorable model to explore inflammatory responses.

Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1–Related Microglial Activation in Neonatal Hypoxic-Ischemic Encephalopathy: Morphologic Consideration

Neonatal hypoxic-ischemic encephalopathy (nHIE) is a major neonatal brain injury. Despite therapeutic hypothermia, mortality and sequelae remain severe. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is associated with the pathophysiology of nHIE. In this study, morphologic change and microglial activation under the nHIE condition and LOX-1 treatment were investigated. The microglial activity and proliferation were assessed with a novel morphologic method, immunostaining, and quantitative PCR in the rat brains of both nHIE model and anti-LOX-1 treatment. Circumference ratio, the long diameter ratio, the cell area ratio, and the roundness of microglia were calculated. The correlation of the morphologic metrics and microglial activation in nHIE model and anti-LOX-1 treated brains was evaluated. LOX-1 was expressed in activated ameboid and round microglia in the nHIE model rat brain. In the evaluation of microglial activation, the novel morphologic metrics correlated with all scales of the nHIE-damaged and treated brains. While the circumference and long diameter ratios had a positive correlation, the cell area ratio and roundness had a negative correlation. Anti-LOX-1 treatment attenuated morphologic microglial activation and proliferation, and suppressed the subsequent production of inflammatory mediators by microglia. In human nHIE, round microglia and endothelial cellsexpressed LOX-1. The results indicate that LOX-1 regulates microglial activation in nHIE and anti-LOX-1 treatment attenuates brain injury by suppressing microglial activation.

Prenatal hypoxemia alters microglial morphology in fetal sheep

ObjectiveNeuroimmune cells, particularly microglia and astrocytes, play a critical role in neurodevelopment. Neurocognitive delays are common in children with congenital heart disease, but their etiology is poorly understood. Our objective was to determine whether prenatal hypoxemia, at levels common in congenital heart disease, induced neuroimmune activation to better understand the origins of neurobehavioral disorders in congenital heart disease. MethodsEight fetal sheep at gestational age 109 ± 3 days (term ∼145 days) were cannulated onto a pumpless extracorporeal oxygenator via the umbilical vessels and supported in a fluid environment for 22 ± 2 days under normoxic (n = 4) or hypoxic (n = 4) conditions. Control fetuses (n = 7) were harvested at gestational age 133 ± 4 days. At necropsy, brains were stained with ionized calcium-binding adaptor molecule 1 and glial fibrillary acidic protein antibodies to quantify microglia and astrocytes, respectively, in gray and white matter in frontotemporal and cerebellar sections. Microglia were classified into 4 morphologic types based on cell shape. Data were analyzed with 1-way analysis of variance or Fisher exact test, as appropriate. ResultsOxygen delivery was significantly reduced in hypoxic fetuses (15.6 ± 1.8 mL/kg/min vs 24.3 ± 2.3 mL/kg/min; P < .01). Rates of apoptosis were similar in hypoxic, normoxic, and intrauterine control animals in all examined areas. There were also no differences between groups in area occupied by glial fibrillary acidic protein-labeled astrocytes or ionized calcium-binding adaptor molecule 1-labeled microglia in all examined areas. However, round microglia were significantly increased in hypoxic animals compared with normoxic animals (33% vs 6%; P < .01) and control animals (33% vs 11%; P < .01). ConclusionsPrenatal hypoxemia altered microglial morphology without significant gliosis. Additional studies characterizing these mechanisms may provide insight into the origins of neurobehavioral disabilities in children with congenital heart disease.

Iba-1-/CD68+ microglia are a prominent feature of age-associated deep subcortical white matter lesions.

Deep subcortical lesions (DSCL) of the brain, are present in ~60% of the ageing population, and are linked to cognitive decline and depression. DSCL are associated with demyelination, blood brain barrier (BBB) dysfunction, and microgliosis. Microglia are the main immune cell of the brain. Under physiological conditions microglia have a ramified morphology, and react to pathology with a change to a more rounded morphology as well as showing protein expression alterations. This study builds on previous characterisations of DSCL and radiologically ‘normal-appearing’ white matter (NAWM) by performing a detailed characterisation of a range of microglial markers in addition to markers of vascular integrity. The Cognitive Function and Ageing Study (CFAS) provided control white matter (WM), NAWM and DSCL human post mortem tissue for immunohistochemistry using microglial markers (Iba-1, CD68 and MHCII), a vascular basement membrane marker (collagen IV) and markers of BBB integrity (fibrinogen and aquaporin 4). The immunoreactive profile of CD68 increased in a stepwise manner from control WM to NAWM to DSCL. This correlated with a shift from small, ramified cells, to larger, more rounded microglia. While there was greater Iba-1 immunoreactivity in NAWM compared to controls, in DSCL, Iba-1 levels were reduced to control levels. A prominent feature of these DSCL was a population of Iba-1-/CD68+ microglia. There were increases in collagen IV, but no change in BBB integrity. Overall the study shows significant differences in the immunoreactive profile of microglial markers. Whether this is a cause or effect of lesion development remains to be elucidated. Identifying microglia subpopulations based on their morphology and molecular markers may ultimately help decipher their function and role in neurodegeneration. Furthermore, this study demonstrates that Iba-1 is not a pan-microglial marker, and that a combination of several microglial markers is required to fully characterise the microglial phenotype.

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.

Microglia increase as photoreceptors decrease in the aging avian retina

Purpose. There is evidence that microglial activation occurs with normal aging in some regions of the brain of rodents. We investigated the pattern of microglia in the retinas of young and aged quail and pigeons to determine if age-related retinal changes evoked migration of microglia into the outer retina. In quail we also investigated the correlation between activated microglia and age-related photo-receptor loss. Methods. Microglia were identified with the monoclonal antibody QH1 in cryosectioned eyes from pigeons, ages 2 to 20 years (n = 14), and in paraffin sections from six-month (n = 15) and one-year-old quail (n = 30). Rounded microglia in the photoreceptor layer were counted in consecutive 400x fields from temporal to nasal. Photoreceptor counts were made from 10 quail retina flat mounts. The photoreceptor number was compared to the number of microglia in corresponding regions of the same retina. Results. Rounded microglia were detected among the photoreceptors of pigeons and quail. Significantly more of these microglia were found in peripheral than in central regions close to the pecten (pigeon p < 0.002 and quail p < 0.01). Furthermore, more microglial cells were present among peripheral photoreceptors of older quail (p < 0.03) and pigeons (p < 0.05) than in the younger birds. In the peripheral retina of the older quail, microglia were significantly and inversely related to the number of photoreceptors (r 2 = 0.9; p < 0.001). Conclusions. Increased microglial were observed in the peripheral retina of both old quail and old pigeons. In the quail, the rounded (activated) microglia were distributed preferentially in regions of greatest photoreceptor loss. Microglial activation does not appear to be a general phenomenon of the aging retina, but in quail activation appears directly related to photoreceptor loss. It is unclear at this time how the change in microglia shape and distribution is related to their neuroprotective/neurotoxic potential.

The effects of pre-natal exposure to methylazoxymethanol acetate on microglia.

The effects of exposure to a cytotoxic agent, methylazoxymethanol acetate (MAM Ac), on the distribution, density and quantitative morphology of microglia in the rat forebrain have been examined with the aid of a peroxidase-conjugated lectin derived from Griffonia simplicifolia. Following exposure to MAM Ac (25 mg/kg maternal body weight) on embryonic day 13 (E13), round microglia were concentrated around the areas of induced cell death at the outer margins of the ventricular germinal zone, particularly in the striatopallidal angle and dorsal thalamus. By E19, there were no detectable differences in microglia distribution between experimental and control animals. The increase in number of microglial cells in the neocortex and caudatoputamen on exposure to MAM Ac lasted for only 4 to 6 days. Subsequently, the number of microglia dropped below control values in both regions. The density of microglia in these areas was similar in control and experimental animals from 6 days after exposure. The proportion of microglia relative to all other cells was also similar at post-natal day 17 (P17) in both experimental and control animals. These results suggest that the distribution and final size of the microglial population is determined by the microenvironment and not by the extent of cell death which may have acted as the initial stimulus to microglial invasion.

Light microscopic study of degenerating cobalt-filled optic axons in goldfish: role of microglia and radial glia in debris removal.

Labeling severed axons with cobaltous-lysine ultimately leads to the degeneration of their distal segments. The present study was designed to determine whether microglia and radial glia have comparable roles in the elimination of degenerating axons. Another purpose was to determine whether the cobalt could escape from degenerating axons and enter intact neuronal cells. Optic axons were filled with cobaltous-lysine for 1 day and the retinal projections were examined from 1 to 106 days later. Optimal filling was obtained 1 day postlabeling. The number of filled axons in the optic tract was significantly reduced at the 2-day time point, indicating that many axons had disintegrated. Many axons contained large swellings that resembled cells. However, transneuronally labeled neuronal cells were never observed. Labeled, rounded microglia appeared among the degenerating axons at the 3-day time point, and the microglia changed shape at 5 days. They became elongated and manifested many processes. In addition, the microglia began to move toward, and entered, the ventricles and vasculature. Virtually all the labeled debris was removed between 17 and 28 days following the application of cobalt. The rapidity with which the axons were removed suggests that the cobalt accelerates the degenerative process either directly, or indirectly by accelerating the arrival of phagocytic cells. Radial glia appeared to play a smaller role in debris elimination. They took up labeled debris to a lesser degree than microglia and were briefly labeled. Interestingly, radial glia did not take up cobalt when it was injected intracranially and diffused through the brain. A previous claim of an axosomatic retinotectal projection to cells deep in the stratum periventriculare of a teleost fish is reinterpreted to represent cobalt within radial glia.