Isolation and Characterization of Ischemia-Derived Astrocytes (IDAs) with Ability to Transactivate Quiescent Astrocytes

Abstract

Reactive gliosis involving activation and proliferation of astrocytes and microglia, is a widespread but largely complex and graded glial response to brain injury. Astroglial population has a previously underestimated high heterogeneity with cells differing in their morphology, gene expression profile, and response to injury. Here, we identified a subset of reactive astrocytes isolated from brain focal ischemic lesions that show several atypical characteristics. Ischemia-derived astrocytes (IDAs) were isolated from early ischemic penumbra and core. IDA did not originate from myeloid precursors, but rather from pre-existing local progenitors. Isolated IDA markedly differ from primary astrocytes, as they proliferate in vitro with high cell division rate, show increased migratory ability, have reduced replicative senescence and grow in the presence of macrophages within the limits imposed by the glial scar. Remarkably, IDA produce a conditioned medium that strongly induced activation on quiescent primary astrocytes and potentiated the neuronal death triggered by oxygen-glucose deprivation. When re-implanted into normal rat brains, eGFP-IDA migrated around the injection site and induced focal reactive gliosis. Inhibition of gamma secretases or culture on quiescent primary astrocytes monolayers facilitated IDA differentiation to astrocytes. We propose that IDA represent an undifferentiated, pro-inflammatory, highly replicative and migratory astroglial subtype emerging from the ischemic microenvironment that may contribute to the expansion of reactive gliosis.Main Points:Ischemia-derived astrocytes (IDA) were isolated from brain ischemic tissueIDA show reduced replicative senescence, increased cell division and spontaneous migrationIDA potentiate death of oxygen-glucose deprived cortical neuronsIDA propagate reactive gliosis on quiescent astrocytes in vitro and in vivoInhibition of gamma secretases facilitates IDA differentiation to astrocytes

Phenotypic heterogeneity of astrocytes in motor neuron disease

“…Table shows some features of aberrant glial cells, including astrocytes or microglia, abnormally‐expressing markers from different cell lineages have been also reported in Alzheimer's disease, Huntington's disease, central nervous system acute lesions and aging, as well as glioma, brain ischemia and trauma, further suggesting the phenotypic switch is strongly associated with inflammation and tissue remodeling after damage.…”

Postischemic Inflammation in Acute Stroke

“…During the first week, the M2 type was found to predominate in the ischemic core while the M1 type predominated in the peri-infarct zone 59. These observations suggest that the peri-infarct region is dominated by proinflammatory and activated microglia whose abundance increases during the first days after ischemic injury 60. The spatial distribution of the microglia phenotypes changes over time, which suggests the enlargement of injured and damaged cerebral tissue.…”

G5G2.5 core‐shell tecto‐dendrimer specifically targets reactive glia in brain ischemia

“…For all surgical procedures animals were anesthetized with ketamine/xylazine (90/10 mg/kg i.p.). Rats were subjected to a unilateral cortical devascularization (CD) as previously described (Herrera and Robertson 1989;Villarreal et al 2011Villarreal et al , 2016 making every effort to reduce the suffering by applying and anesthetic mixture that has analgesic effects (Lee-Parritz 2007) and the number of animals used was reduced by the appropriate calculations (see below) and using the contralateral non-ischemic hemisphere as internal control in each animal. Briefly, rats were placed in a stereotaxic frame so that the head was held in place during the procedure, and after dissecting the temporal muscle and the fascia, a small 2 9 2 mm surface of the skull between the coronal suture and the bregma line was removed, exposing the underlying vasculature.…”

“…Analysis was made in blind conditions on coded images. Experiments involving animals were performed independently three times, and each experiment used at least three animals based in the past experience with the animal model (Villarreal et al 2014(Villarreal et al , 2016 and sample size calculations by resource equation (Charan and Kantharia 2013). This calculation rendered a total number of 10 animals as necessary for these experiments since only one group was defined, with an unique dose and being the internal control the contralateral hemisphere in each animal.…”

“…G5G2.5 tecto-dendrimer reaches glial cells in the ischemic brain Experimental models of focal ischemia in rodents reproduce most of the clinical features found in ischemic human brains. Unilateral CD induces a permanent cortical focal ischemia with a clearly defined ischemic core and surrounding penumbra (Villarreal et al 2014(Villarreal et al , 2016. At 3 days postischemic lesion (DPL), the ischemic core presents a GFAPnegative staining, and is invaded by blood-borne macrophages cells, whereas the surrounding penumbral area shows hypertrophied GFAP+ reactive glia.…”