Microglial precursors derived from mouse embryonic stem cells.

Microglia are resident immune cells of the central nervous system. They can be directly isolated from the brain or from mixed postnatal glial cultures. Isolation of primary microglia is inefficient due to low yield. The cell line BV2 was used as a substitute for primary microglia, but BV2 are oncogenically transformed cells. Here, we established a protocol to generate microglial precursor lines from mouse embryonic stem (ES) cells. Microglial precursor cells were obtained from murine ES cells by differentiation of embryoid bodies to microglia within a mixed brain culture. Several independent ES cell-derived microglial precursor (ESdM) lines were generated and characterized by flow cytometry, immunocytochemistry, and functional assays. All ESdM showed expression of IBa1, CD11b, CD45, F4/80, CD49d, and CD29, but were negative for cKit and CD34. Stimulation with interferon-gamma or lipopolysaccharide (LPS) demonstrated upregulation of proinflammatory cytokine gene transcription including nitric oxide synthase-2, interleukin-1beta, and tumor necrosis factor-alpha at levels comparable to primary microglia. The ESdM showed efficient and rapid phagocytosis of microsphere beads, which was increased after stimulation with LPS. ESdM expressed the chemokine receptor CX3CR1 and demonstrated directed migration toward the ligand CX3CL1. After in vivo transplantation into postnatal brain tissue, ESdM showed engraftment as cells with a microglial phenotype and morphology. Thus, ESdM are stable proliferating cells substantially having most characteristics of primary microglia and therefore being a suitable tool to study microglial function in vitro and in vivo.

Prenatal choline supplementation in rats increases the expression of IGF2 and its receptor IGF2R and enhances IGF2-induced acetylcholine release in hippocampus and frontal cortex.

Choline is an essential nutrient whose availability during the second half of gestation produces long-lasting cognitive effects. Rats that obtain supplemental choline during embryonic day (E) 11-17 have enhanced depolarization-evoked acetylcholine (ACh) release from hippocampal slices, whereas choline deficiency during this time reduces this release. Previously we reported that rats whose mothers consumed a choline-supplemented diet during E11-17 have higher levels of insulin-like growth factor II (IGF2) mRNA and protein in the frontal cortex compared to control and prenatally choline-deficient animals. Since IGF2 has been shown to stimulate endogenous ACh release, we measured the release of ACh from hippocampal and frontal cortical slices from rats on postnatal day (P) 18, P24, P34 and P80 in response to a depolarizing concentration of potassium (45 mM or 25 mM) or to IGF2 treatment in the absence or presence of a depolarizing concentration of potassium (25 mM). On P18, IGF2/depolarization-evoked ACh release from hippocampal slices was enhanced by prenatal choline supplementation. In the frontal cortex on P80, prenatal choline supplementation dramatically potentiated ACh release induced by depolarization, IGF2 or the combination of the two. On P18 and P90 and in both brain regions, IGF2 mRNA and protein levels, as well as protein levels of the IGF2 receptor (IGF2R), were higher in prenatally choline-supplemented rats. Choline supplementation also increased IGF2R mRNA levels in the septum. In summary, prenatal choline supplementation produced alterations in IGF2 signaling, via increased levels of IGF2 and IGF2R, which may enhance cholinergic neurotransmission and confer neuroprotection against insult.

Protective effects of microglia in multiple sclerosis.

The role of microglia in demyelinating neurodegenerative diseases such as multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) is still controversial. Although microglial cells are known as the professional phagocytes and executer of innate immunity in the central nervous system (CNS), it is believed that microglia are rather neurotoxic in these diseases. However, there is recent evidence indicating that microglia could also exert a neuroprotective function in MS and EAE. First evidence for the protective effect of immune cells in CNS diseases emerged from studies in invertebrates. In the medicinal leech, the process of regeneration begins with rapid activation and accumulation of phagocytic glial cells at the lesion site followed by phagocytosis of damaged tissue by these cells which promoted robust neural regeneration. In vertebrates, several lines of evidence demonstrate that microglia are also involved in neuroprotection by the secretion of soluble mediators that trigger neural repair and usually contribute to the creation of an environment conductive for regeneration. The efficient removal of apoptotic cells and clearance of debris at the lesion site and the recruitment of stem cell populations as well as the induction of neurogenesis are directly correlated. These findings suggest that microglia play a major role in creating a microenvironment for repair and regenerative processes in demyelinating neuroinflammatory diseases.

Generation of microglial cells from mouse embryonic stem cells.

Microglia, the resident immune cells of the brain, are difficult to obtain in high numbers and purity using currently available methods; to date, microglia for experimental research are mainly isolated from the brain or from mixed glial cultures. In this paper, we describe a basic protocol for the in vitro differentiation of mouse embryonic stem (ES) cells into microglial precursor cells. Microglia are obtained by a protocol consisting of five stages: (i) cultivation of ES cells, (ii) formation and differentiation of embryoid bodies, (iii) differentiation into neuroectodermal lineage and isolation of myeloid precursor cells, (iv) differentiation into microglial precursor cells and (v) cultivation of ES cell-derived microglial precursors (ESdMs). The protocol can be completed in 60 d and results in stably proliferating ESdM lines, which show inducible transcription of inflammatory genes and cell marker expression comparable with primary microglia. Furthermore, ESdMs are capable of chemokine-directed migration and phagocytosis, which are major functional features of microglia.

Perfil clínico-demográfico de idosos em internamento domiciliar / The clinic and demographic characteristics of aged patients interned in a home care

Este trabalho trata de um estudo observacional, descritivo e retrospectivo, que teve como objetivo identificar características clínicas e demográficas de pacientes idosos internados em uma empresa de internamento domiciliar, localizada na cidade de Salvador, Bahia, no recorte temporal entre agosto e outubro de 2000. Observou-se que a maioria desses pacientes teve como diagnóstico as doenças vascuolares, com modalidade de assistência de seis horas e dependência parcial da enfermagem.