Gravity plays a crucial role in physiology. The lack of gravity, like in long duration spaceflight missions, cause pathologies in
e.g.
, the musculoskeletal system, cardiovascular deconditioning, ...immune system deprivation or brain abnormalities, to just mention a few. The application of artificial gravity through short-arm human centrifugation (SAHC) has been studied as a possible countermeasure to treat spaceflight deconditioning. However, hypergravity protocols applied by using SAHC have also been used to treat different, ground-based pathologies. Such gravitational therapies have been applied in Uruguay for more than four decades now. The aim of this overview is to summarize the most important findings about the effects of gravitational therapy in different, mainly vascular based pathologies according to the experience in the Gravitational Therapy Center and to discuss the current research in the field of hypergravity applications in medicine but also as multisystem countermeasure for near weightlessness pathologies. New insight is needed on the use of hypergravity in medicine and space research and application.
Perivascular pericytes are key regulators of the blood–brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, ...pericyte identification remains challenging. A previous study reported that the far‐red fluorophore TO‐PRO‐3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO‐PRO‐3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO‐PRO‐3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron‐glial antigen 2 (NG2) and platelet‐derived growth factor receptor beta antigen (PDGFrβ) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO‐PRO‐3 signal enabled quantification of pericytes density and morphometry; likewise, TO‐PRO‐3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO‐PRO‐3 stained cells expressed the contractile protein α–SMA, indicative of their ability to control the capillary diameter. Uptake of TO‐PRO‐3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein‐mediated active transport sustained dye incorporation. We conclude that TO‐PRO‐3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature.
Identification of capillary pericytes remains challenging. The nuclear far‐red fluorophore TO‐PRO‐3 was previously employed to label the pericytes of the subventricular zone (Lacar et al., 2012). Herein, we show that ex vivo living pericytes from the hippocampus, cortex, spinal cord, and retina selectively uptake TO‐PRO‐3. Uptake involves a transporter that actively concentrates the dye into pericytes allowing their identification. The good stability, brightness, affordability, and reproducibility of the fluorescent signal, together with the ease of the labeling procedure, makes TO‐PRO‐3 a convenient method to trace cerebral pericytes during multiple labeling circumventing the need for reporter mice or immunohistochemical counter‐staining.
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive death of motor neurons and muscle atrophy, with defective neuron-glia interplay and emergence of ...aberrant glial phenotypes having a role in disease pathology. Here, we have studied if the pigment violacein with several reported protective/antiproliferative properties may control highly neurotoxic astrocytes (AbAs) obtained from spinal cord cultures of symptomatic hSOD1G93A rats, and if it could be neuroprotective in this ALS experimental model. At concentrations lower than those reported as protective, violacein selectively killed aberrant astrocytes. Treatment of hSOD1G93A rats with doses equivalent to the concentrations that killed AbAs caused a marginally significant delay in survival, partially preserved the body weight and soleus muscle mass and improved the integrity of the neuromuscular junction. Reduced motor neuron death and glial reactivity was also found and likely related to decreased inflammation and matrix metalloproteinase-2 and -9. Thus, in spite that new experimental designs aimed at extending the lifespan of hSOD1G93A rats are needed, improvements observed upon violacein treatment suggest a significant therapeutic potential that deserves further studies.
In the rat model of amyotrophic lateral sclerosis expressing the G93A superoxide dismutase-1 mutation, motor neuron death and rapid paralysis progression are associated with the emergence of a ...population of aberrant glial cells (AbAs) that proliferate in the degenerating spinal cord. Targeting of AbAs with anti-neoplasic drugs reduced paralysis progression, suggesting a pathogenic potential contribution of these cells accelerating paralysis progression. In the present study, analyze the cellular and ultrastructural features of AbAs following their isolation and establishment in culture during several passages. We found that AbAs exhibit permanent loss of contact inhibition, absence of intermediate filaments and abundance of microtubules, together with an important production of extracellular matrix components. Remarkably, AbAs also exhibited exacerbated ER stress together with a significant abundance of lipid droplets, as well as autophagic and secretory vesicles, all characteristic features of cellular stress and inflammatory activation. Taken together, the present data show AbA cells as a unique aberrant phenotype for a glial cell that might explain their pathogenic and neurotoxic effects.
In amyotrophic lateral sclerosis (ALS), astrocytes are considered key players in some non-cell non-neuronal autonomous mechanisms that underlie motor neuron death. However, it is unknown how much of ...these deleterious features were permanently acquired. To assess this point, we evaluated if the most remarkable features of neurotoxic aberrant glial phenotypes (AbAs) isolated from paralytic rats of the ALS model G93A Cu/Zn superoxide dismutase 1 (SOD1) could remain upon long lasting cultivation. Real time PCR, immunolabelling and zymography analysis showed that upon many passages, AbAs preserved the cell proliferation capacity, mitochondrial function and response to different compounds that inhibit some key astrocyte functions but decreased the expression of parameters associated to cell lineage, homeostasis and inflammation. As these results are contrary to the sustained inflammatory status observed along disease progression in SOD1G93A rats, we propose that the most AbAs remarkable features related to homeostasis and neurotoxicity were not permanently acquired and might depend on the signaling coming from the injuring microenvironment present in the degenerating spinal cord of terminal rats.
This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PRO
-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with ...TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.
The white matter is an important constituent of the central nervous system, containing axons, oligodendrocytes, and its progenitor cells, astrocytes, and microglial cells. Oligodendrocytes are ...central for myelin synthesis, the insulating envelope that protects axons and allows normal neural conduction. Both, oligodendrocytes and myelin, are highly vulnerable to toxic factors in many neurodevelopmental and neurodegenerative disorders associated with disturbances of myelination. Here we review the main alterations in oligodendrocytes and myelin observed in some organic acidurias/acidemias, which correspond to inherited neurometabolic disorders biochemically characterized by accumulation of potentially neurotoxic organic acids and their derivatives. The yet incompletely understood mechanisms underlying the high vulnerability of OLs and/or myelin in glutaric acidemia type I, the most prototypical cerebral organic aciduria, are particularly discussed.
Iron deficiency anemia is a prevalent health problem among pregnant women and infants, particularly in the developing countries that causes brain development deficits and poor cognitive outcomes. ...Since tissue iron depletion may impair myelination and trigger cellular hypoxic signaling affecting blood vessels, we studied myelination and the neurovascular unit (NVU) in infant rats born to mothers fed with an iron deficient (ID) or control diet from embryonic day 5 till weaning. Blood samples and brains of rat pups at postnatal day (PND) 14 and 30 were analyzed. PND 14 ID rats had severe microcytic hypochromic anemia that was almost reversed at PND 30 although hypomyelination and astrocyte immature phenotype in the corpus callosum were significant at that age. In CA1 hippocampal region, PND 14 and PND 30 ID rats showed significant reduced expression of the receptor β of the platelet-derived growth factor localized in pericytes and associated to aquaporin 4 (AQP4) immunopositive capillaries. Shorter AQP4 + capillaries and reduced AQP4 expression were also evidenced in PND 14 and PND 30 ID rats. In addition, pericyte membrane permeability through large-pore channels was transiently increased in ID rats at PND 14 but not at PND 30, while the blood–brain barrier permeability was not affected. Remarkably, transient increased pericyte permeability found in PND 14 ID rats was not directly related to iron depletion, suggesting the involvement of other iron deficiency anemia-induced mechanisms. In summary, severe ID during gestation and lactation produces persistent hypomyelination and significantly affects hippocampal pericytes and astrocytes in the NVU which may trigger impaired neurovascular function.
Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid ...(GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 μm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.
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