Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Background: The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin(+) Sox2(+) neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium). -- Results: Here we report the isolation and long term propagation of another population of Nestin(+) cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges. -- Conclusion: Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

GFAP Isoforms in Adult Mouse Brain with a Focus on Neurogenic Astrocytes and Reactive Astrogliosis in Mouse Models of Alzheimer Disease

24 p.

Unlike Physical Exercise, Modified Environment Increases the Lifespan of SOD1(G93A) Mice However Both Conditions Induce Cellular Changes

8 p.

GABA release by hippocampal astrocytes

10 p.

Early astrocytic atrophy in the entorhinal cortex of a triple transgenic animal model of Alzheimer's disease

The EC (entorhinal cortex) is fundamental for cognitive and mnesic functions. Thus damage to this area appears as a key element in the progression of AD (Alzheimer's disease), resulting in memory deficits arising from neuronal and synaptic alterations as well as glial malfunction. In this paper, we have performed an in-depth analysis of astroglial morphology in the EC by measuring the surface and volume of the GFAP (glial fibrillary acidic protein) profiles in a triple transgenic mouse model of AD [3xTg-AD (triple transgenic mice of AD)]. We found significant reduction in both the surface and volume of GFAP-labelled profiles in 3xTg-AD animals from very early ages (1 month) when compared with non-Tg (non-transgenic) controls (48 and 54%, reduction respectively), which was sustained for up to 12 months (33 and 45% reduction respectively). The appearance of Lambda beta (amyloid beta-peptide) depositions at 12 months of age did not trigger astroglial hypertrophy; nor did it result in the close association of astrocytes with senile plaques. Our results suggest that the AD progressive cognitive deterioration can be associated with an early reduction of astrocytic arborization and shrinkage of the astroglial domain, which may affect synaptic connectivity within the EC and between the EC and other brain regions. In addition, the EC seems to be particularly vulnerable to AD pathology because of the absence of evident astrogliosis in response to A beta accumulation. Thus we can consider that targeting astroglial atrophy may represent a therapeutic strategy which might slow down the progression of AD.

Increase in the density of resting microglia precedes neuritic plaque formation and microglial activation in a transgenic model of Alzheimer's disease

The formation of cerebral senile plaques composed of amyloid beta peptide (A beta) is a fundamental feature of Alzheimer's disease (AD). Glial cells and more specifically microglia become reactive in the presence of A beta. In a triple transgenic model of AD (3 x Tg-AD), we found a significant increase in activated microglia at 12 (by 111%) and 18 (by 88%) months of age when compared with non-transgenic (non-Tg) controls. This microglial activation correlated with A beta plaque formation, and the activation in microglia was closely associated with A beta plaques and smaller A beta deposits. We also found a significant increase in the area density of resting microglia in 3 x Tg-AD animals both at plaque-free stage (at 9 months by 105%) and after the development of A plaques (at 12 months by 54% and at 18 months by 131%). Our results show for the first time that the increase in the density of resting microglia precedes both plaque formation and activation of microglia by extracellular A beta accumulation. We suggest that AD pathology triggers a complex microglial reaction: at the initial stages of the disease the number of resting microglia increases, as if in preparation for the ensuing activation in an attempt to fight the extracellular A beta load that is characteristic of the terminal stages of the disease. Cell Death and Disease (2010) 1, e1; doi:10.1038/cddis.2009.2; published online 14 January 2010

Cytokine pathway disruption in a mouse model of schizophrenia induced by Munc18-1a overexpression in the brain

Background: An accumulating body of evidence points to the significance of neuroinflammation and immunogenetics in schizophrenia, and an imbalance of cytokines in the central nervous system (CNS) has been suggested to be associated with the disorder. Munc18-overexpressing mice (Munc18-OE) have provided a model for the study of the alterations that may underlie the symptoms of subjects with schizophrenia. The aim of the present study was to elucidate the involvement of neuroinflammation and cytokine imbalance in this model. Methods: Cytokines were evaluated in the cortex and the striatum of Munc18-OE and wild-type (WT) mice by enzyme-linked immunosorbent assay (ELISA). Protein levels of specific microglia and macrophage, astrocytic and neuroinflammation markers were quantified by western blot in the cortex and the striatum of Munc18-OE and WT mice. Results: Each cytokine evaluated (Interferon-gamma (IFN-gamma), Tumor Necrosis Factor-alpha (TNF-alpha), Interleukin-2 (IL-2) and CCL2 chemokine) was present at higher levels in the striatum of Munc18-OE mice than WT. Cortical TNF-alpha and IL-2 levels were significantly lower in Munc18-OE mice than WT mice. The microglia and macrophage marker CD11b was lower in the cortexes of Munc18-OE mice than WT, but no differences were observed in the striatum. Glial Fibrillary Acidic Protein (GFAP) and Nuclear Factor-kappaB (NF-kappa B)p65 levels were not different between the groups. Interleukin-1beta (IL-1 beta) and IL-6 levels were beneath detection limits. Conclusions: The disrupted levels of cytokines detected in the brain of Munc18-OE mice was found to be similar to clinical reports and endorses study of this type for analysis of this aspect of the disorder. The lower CD11b expression in the cortex but not in the striatum of the Munc18-OE mice may reflect differences in physiological activity. The cytokine expression pattern observed in Munc18-OE mice is similar to a previously published model of schizophrenia caused by maternal immune activation. Together, these data suggest a possible role for an immune imbalance in this disorder.

Increased antiparkinson efficacy of the combined administration of VEGF- and GDNF-loaded nanospheres in a partial lesion model of Parkinson's disease

Current research efforts are focused on the application of growth factors, such as glial cell line-derived neurotrophic factor (GDNF) and vascular endothelial growth factor (VEGF), as neuroregenerative approaches that will prevent the neurodegenerative process in Parkinson's disease. Continuing a previous work published by our research group, and with the aim to overcome different limitations related to growth factor administration, VEGF and GDNF were encapsulated in poly(lactic-co-glycolic acid) nanospheres (NS). This strategy facilitates the combined administration of the VEGF and GDNF into the brain of 6-hydroxydopamine (6-OHDA) partially lesioned rats, resulting in a continuous and simultaneous drug release. The NS particle size was about 200 nm and the simultaneous addition of VEGF NS and GDNF NS resulted in significant protection of the PC-12 cell line against 6-OHDA in vitro. Once the poly(lactic-co-glycolic acid) NS were implanted into the striatum of 6-OHDA partially lesioned rats, the amphetamine rotation behavior test was carried out over 10 weeks, in order to check for in vivo efficacy. The results showed that VEGF NS and GDNF NS significantly decreased the number of amphetamine-induced rotations at the end of the study. In addition, tyrosine hydroxylase immunohistochemical analysis in the striatum and the external substantia nigra confirmed a significant enhancement of neurons in the VEGF NS and GDNF NS treatment group. The synergistic effect of VEGF NS and GDNF NS allows for a reduction of the dose by half, and may be a valuable neurogenerative/neuroreparative approach for treating Parkinson's disease.

Pio del Rio Hortega and the discovery of the oligodendrocytes

Rio del Rio Hortega (1882-1945) discovered microglia and oligodendrocytes (OLGs), and after Ramon y Cajal, was the most prominent figure of the Spanish school of neurology. He began his scientific career with Nicolas Achucarro from whom he learned the use of metallic impregnation techniques suitable to study non-neuronal cells. Later on, he joined Cajal's laboratory. and Subsequently, he created his own group, where he continued to develop other innovative modifications of silver staining methods that revolutionized the study of glial cells a century ago. He was also interested in neuropathology and became a leading authority on Central Nervous System (CNS) tumors. In parallel to this clinical activity, del Rio Hortega rendered the first systematic description of a major polymorphism present in a subtype of macroglial cells that he named as oligodendroglia and later OLGs. He established their ectodermal origin and suggested that they built the myelin sheath of CNS axons, just as Schwann cells did in the periphery. Notably, he also suggested the trophic role of OLGs for neuronal functionality, an idea that has been substantiated in the last few years. Del Rio Hortega became internationally recognized and established an important neurohistological school with outstanding pupils from Spain and abroad, which nearly disappeared after his exile due to the Spanish civil war. Yet, the difficulty of metal impregnation methods and their variability in results, delayed for some decades the confirmation of his great insights into oligodendrocyte biology until the development of electron microscopy and immunohistochemistry. This review aims at summarizing the pioneer and essential contributions of del Rio Hortega to the current knowledge of oligodendrocyte structure and function, and to provide a hint of the scientific personality of this extraordinary and insufficiently recognized man.

From the Cajal alumni Achucarro and Rio-Hortega to the rediscovery of never-resting microglia

Under the guidance of Ramon y Cajal, a plethora of students flourished and began to apply his silver impregnation methods to study brain cells other than neurons: the neuroglia. In the first decades of the twentieth century, Nicolas Achucarro was one of the first researchers to visualize the brain cells with phagocytic capacity that we know today as microglia. Later, his pupil Pio del Rio-Hortega developed modifications of Achucarro's methods and was able to specifically observe the fine morphological intricacies of microglia. These findings contradicted Cajal's own views on cells that he thought belonged to the same class as oligodendroglia (the so called "third element" of the nervous system), leading to a long-standing discussion. It was only in 1924 that Rio-Hortega's observations prevailed worldwide, thus recognizing microglia as a unique cell type. This late landing in the Neuroscience arena still has repercussions in the twenty first century, as microglia remain one of the least understood cell populations of the healthy brain. For decades, microglia in normal, physiological conditions in the adult brain were considered to be merely "resting," and their contribution as "activated" cells to the neuroinflammatory response in pathological conditions mostly detrimental. It was not until microglia were imaged in real time in the intact brain using two-photon in vivo imaging that the extreme motility of their fine processes was revealed. These findings led to a conceptual revolution in the field: "resting" microglia are constantly surveying the brain parenchyma in normal physiological conditions. Today, following Cajal's school of thought, structural and functional investigations of microglial morphology, dynamics, and relationships with neurons and other glial cells are experiencing a renaissance and we stand at the brink of discovering new roles for these unique immune cells in the healthy brain, an essential step to understand their causal relationship to diseases.