Increased Astrocyte Proliferation Research Articles

Hyperglycemia enhances brain susceptibility to lipopolysaccharide-induced neuroinflammation via astrocyte reprogramming.

Hyperglycemia has been shown to modulate the immune response of peripheral immune cells and organs, but the impact of hyperglycemia on neuroinflammation within the brain remains elusive. In the present study, we provide evidences that streptozotocin (STZ)-induced hyperglycemic condition in mice drives a phenotypic switch of brain astrocytes to a proinflammatory state, and increases brain vulnerability to mild peripheral inflammation. In particular, we found that hyperglycemia led to a significant increase in the astrocyte proliferation as determined by flow cytometric and immunohistochemical analyses of mouse brain. The increased astrocyte proliferation by hyperglycemia was reduced by Glut1 inhibitor BAY-876. Transcriptomic analysis of isolated astrocytes from Aldh1l1CreERT2;tdTomato mice revealed that peripheral STZ injection induced astrocyte reprogramming into proliferative, and proinflammatory phenotype. Additionally, STZ-induced hyperglycemic condition significantly enhanced the infiltration of circulating myeloid cells into the brain and the disruption of blood-brain barrier in response to mild lipopolysaccharide (LPS) administration. Systemic hyperglycemia did not alter the intensity and sensitivity of peripheral inflammation in mice to LPS challenge, but increased the inflammatory potential of brain microglia. In line with findings from mouse experiments, a high-glucose environment intensified the LPS-triggered production of proinflammatory molecules in primary astrocyte cultures. Furthermore, hyperglycemic mice exhibited a significant impairment in cognitive function after mild LPS administration compared to normoglycemic mice as determined by novel object recognition and Y-maze tasks. Taken together, these results demonstrate that hyperglycemia directly induces astrocyte reprogramming towards a proliferative and proinflammatory phenotype, which potentiates mild LPS-triggered inflammation within brain parenchymal regions.

Isoflavones Mediate Dendritogenesis Mainly through Estrogen Receptor α.

The nuclear estrogen receptor (ER) and G-protein-coupled ER (GPER1) play a crucial role during brain development and are involved in dendrite and spine growth as well as synapse formation. Soybean isoflavones, such as genistein, daidzein, and S-equol, a daidzein metabolite, exert their action through ER and GPER1. However, the mechanisms of action of isoflavones on brain development, particularly during dendritogenesis and neuritogenesis, have not yet been extensively studied. We evaluated the effects of isoflavones using mouse primary cerebellar culture, astrocyte-enriched culture, Neuro-2A clonal cells, and co-culture with neurons and astrocytes. Soybean isoflavone-augmented estradiol mediated dendrite arborization in Purkinje cells. Such augmentation was suppressed by co-exposure with ICI 182,780, an antagonist for ERs, or G15, a selective GPER1 antagonist. The knockdown of nuclear ERs or GPER1 also significantly reduced the arborization of dendrites. Particularly, the knockdown of ERα showed the greatest effect. To further examine the specific molecular mechanism, we used Neuro-2A clonal cells. Isoflavones also induced neurite outgrowth of Neuro-2A cells. The knockdown of ERα most strongly reduced isoflavone-induced neurite outgrowth compared with ERβ or GPER1 knockdown. The knockdown of ERα also reduced the mRNA levels of ER-responsive genes (i.e., Bdnf, Camk2b, Rbfox3, Tubb3, Syn1, Dlg4, and Syp). Furthermore, isoflavones increased ERα levels, but not ERβ or GPER1 levels, in Neuro-2A cells. The co-culture study of Neuro-2A cells and astrocytes also showed an increase in isoflavone-induced neurite growth, and co-exposure with ICI 182,780 or G15 significantly reduced the effects. In addition, isoflavones increased astrocyte proliferation via ER and GPER1. These results indicate that ERα plays an essential role in isoflavone-induced neuritogenesis. However, GPER1 signaling is also necessary for astrocyte proliferation and astrocyte-neuron communication, which may lead to isoflavone-induced neuritogenesis.

HIV Nef Expression Down-modulated GFAP Expression and Altered Glutamate Uptake and Release and Proliferation in Astrocytes.

HIV infection of astrocytes leads to restricted gene expression and replication but abundant expression of HIV early genes Tat, Nef and Rev. A great deal of neuroHIV research has so far been focused on Tat protein, its effects on astrocytes, and its roles in neuroHIV. In the current study, we aimed to determine effects of Nef expression on astrocytes and their function. Using transfection or infection of VSVG-pseudotyped HIV viruses, we showed that Nef expression down-modulated glial fibrillary acidic protein (GFAP) expression. We then showed that Nef expression also led to decreased GFAP mRNA expression. The transcriptional regulation was further confirmed using a GFAP promoter-driven reporter gene assay. We performed transcription factor profiling array to compare the expression of transcription factors between Nef-intact and Nef-deficient HIV-infected cells and identified eight transcription factors with expression changes of 1.5-fold or higher: three up-regulated by Nef (Stat1, Stat5, and TFIID), and five down-regulated by Nef (AR, GAS/ISRE, HIF, Sp1, and p53). We then demonstrated that removal of the Sp1 binding sites from the GFAP promoter resulted in a much lower level of the promoter activity and reversal of Nef effects on the GFAP promoter, confirming important roles of Sp1 in the GFAP promoter activity and for Nef-induced GFAP expression. Lastly, we showed that Nef expression led to increased glutamate uptake and decreased glutamate release by astrocytes and increased astrocyte proliferation. Taken together, these results indicate that Nef leads to down-modulation of GFAP expression and alteration of glutamate metabolism in astrocytes, and astrocyte proliferation and could be an important contributor to neuroHIV.

Perinatal Hypoxemia and Oxygen Sensing.

The development of the control of breathing begins in utero and continues postnatally. Fetal breathing movements are needed for establishing connectivity between the lungs and central mechanisms controlling breathing. Maturation of the control of breathing, including the increase of hypoxia chemosensitivity, continues postnatally. Insufficient oxygenation, or hypoxia, is a major stressor that can manifest for different reasons in the fetus and neonate. Though the fetus and neonate have different hypoxia sensing mechanisms and respond differently to acute hypoxia, both responses prevent deviations to respiratory and other developmental processes. Intermittent and chronic hypoxia pose much greater threats to the normal developmental respiratory processes. Gestational intermittent hypoxia, due to maternal sleep-disordered breathing and sleep apnea, increases eupneic breathing and decreases the hypoxic ventilatory response associated with impaired gasping and autoresuscitation postnatally. Chronic fetal hypoxia, due to biologic or environmental (i.e. high-altitude) factors, is implicated in fetal growth restriction and preterm birth causing a decrease in the postnatal hypoxic ventilatory responses with increases in irregular eupneic breathing. Mechanisms driving these changes include delayed chemoreceptor development, catecholaminergic activity, abnormal myelination, increased astrocyte proliferation in the dorsal respiratory group, among others. Long-term high-altitude residents demonstrate favorable adaptations to chronic hypoxia as do their offspring. Neonatal intermittent hypoxia is common among preterm infants due to immature respiratory systems and thus, display a reduced drive to breathe and apneas due to insufficient hypoxic sensitivity. However, ongoing intermittent hypoxia can enhance hypoxic sensitivity causing ventilatory overshoots followed by apnea; the number of apneas is positively correlated with degree of hypoxic sensitivity in preterm infants. Chronic neonatal hypoxia may arise from fetal complications like maternal smoking or from postnatal cardiovascular problems, causing blunting of the hypoxic ventilatory responses throughout at least adolescence due to attenuation of carotid body fibers responses to hypoxia with potential roles of brainstem serotonin, microglia, and inflammation, though these effects depend on the age in which chronic hypoxia initiates. Fetal and neonatal intermittent and chronic hypoxia are implicated in preterm birth and complicate the respiratory system through their direct effects on hypoxia sensing mechanisms and interruptions to the normal developmental processes. Thus, precise regulation of oxygen homeostasis is crucial for normal development of the respiratory control network. © 2021 American Physiological Society. Compr Physiol 11:1653-1677, 2021.

Effect of fractionated whole-brain irradiation on brain and plasma in a rat model: Metabolic, volumetric and histopathological changes

In the present study, we investigated the correlation between histopathological, metabolic, and volumetric changes in the brain and plasma under experimental conditions. Adult male Wistar rats received fractionated whole-brain irradiation (fWBI) with a total dose of 32 Gy delivered in 4 fractions (dose 8 Gy per fraction) once a week on the same day for 4 consecutive weeks. Proton magnetic resonance spectroscopy (1H MRS) and imaging were used to detect metabolic and volumetric changes in the brain and plasma. Histopathological changes in the brain were determined by image analysis of immunofluorescent stained sections. Metabolic changes in the brain measured by 1H MRS before, 48 h, and 9 weeks after the end of fWBI showed a significant decrease in the ratio of total N-acetylaspartate to total creatine (tNAA/tCr) in the corpus striatum. We found a significant decrease in glutamine + glutamate/tCr (Glx/tCr) and, conversely, an increase in gamma-aminobutyric acid to tCr (GABA/tCr) in olfactory bulb (OB). The ratio of astrocyte marker myoinositol/tCr (mIns/tCr) significantly increased in almost all evaluated areas. Magnetic resonance imaging (MRI)-based brain volumetry showed a significant increase in volume, and a concomitant increase in the T2 relaxation time of the hippocampus. Proton nuclear magnetic resonance (1H NMR) plasma metabolomics displayed a significant decrease in the level of glucose and glycolytic intermediates and an increase in ketone bodies. The histomorphological analysis showed a decrease to elimination of neuroblasts, increased astrocyte proliferation, and a mild microglia response. The results of the study clearly reflect early subacute changes 9–11 weeks after fWBI with strong manifestations of brain edema, astrogliosis, and ongoing ketosis.

Cross‐talk between monocyte invasion and astrocyte proliferation regulates scarring in brain injury

Scar formation after brain injury is still poorly understood. To further elucidate such processes, here, we examine the interplay between astrocyte proliferation taking place predominantly at the vascular interface and monocyte invasion. Using genetic mouse models that decrease or increase reactive astrocyte proliferation, we demonstrate inverse effects on monocyte numbers in the injury site. Conversely, reducing monocyte invasion using CCR2−/− mice causes a strong increase in astrocyte proliferation, demonstrating an intriguing negative cross‐regulation between these cell types at the vascular interface. CCR2−/− mice show reduced scar formation with less extracellular matrix deposition, smaller lesion site and increased neuronal coverage. Surprisingly, the GFAP+ scar area in these mice is also significantly decreased despite increased astrocyte proliferation. Proteomic analysis at the peak of increased astrocyte proliferation reveals a decrease in extracellular matrix synthesizing enzymes in the injury sites of CCR2−/− mice, highlighting how early key aspects of scar formation are initiated. Taken together, we provide novel insights into the cross‐regulation of juxtavascular proliferating astrocytes and invading monocytes as a crucial mechanism of scar formation upon brain injury.

2,3,5,4'-Tetrahydroxystilbene-2-O-beta-D-glucoside Reverses Stress-Induced Depression via Inflammatory and Oxidative Stress Pathways.

Major depressive disorder (MDD) is a chronic mental disease that adversely affects human mood and cognition. Many first-line antidepressant drugs have high rates of partial responsiveness or nonresponsiveness with side effects, and finding more effective drugs for the treatment of depression is therefore urgently needed. THSG, a main active compound of the traditional Chinese herb Polygonum multiflorum, reportedly acts as a neuroprotective agent. This study aimed to illustrate whether THSG prevents depressive-like behaviors induced by chronic restraint stress (CRS) in an MDD mouse model. Our results demonstrated that the peripheral administration of different THSG doses (10 mg/kg, 20 mg/kg, and 40 mg/kg) reversed the depressive-like behaviors in CRS mice as measured by the tail suspension test, forced swimming test, and open-field test. Further analyses suggested that THSG treatment reduced oxidative stress in both the central and peripheral nervous systems of CRS mice. In addition, heightened inflammatory responses, demonstrated by the increased expression of proinflammatory factors (TNF-α, IL-1β, and IL-6), in hippocampal and prefrontal cortex tissues of CRS mice were inhibited by THSG administration. THSG also restored the diminished Akt signaling pathway in the brains of CRS mice. Moreover, our data suggest increased astrocyte proliferation and neurogenesis in the hippocampus of CRS mice after THSG treatment. Taken together, our results demonstrated an antidepressant effect of THSG in a mouse model of MDD for the first time, and oxidative stress and inflammatory pathways were determined to play roles in this effect.

The neuroprotective effects of preconditioning exercise on brain damage and neurotrophic factors after focal brain ischemia in rats

Preconditioning exercise can exert neuroprotective effects after stroke. However, the mechanism underlying these neuroprotective effects by preconditioning exercise remains unclear. We investigated the neuroprotective effects of preconditioning exercise on brain damage and the expression levels of the midkine (MK) and brain-derived neurotrophic factor (BDNF) after brain ischemia. Animals were assigned to one of 4 groups: exercise and ischemia (Ex), no exercise and ischemia (No-Ex), exercise and no ischemia (Ex-only), and no exercise and intact (Control). Rats ran on a treadmill for 30min once a day at a speed of 25m/min for 5days a week for 3 weeks. After the exercise program, stroke was induced by a 60min left middle cerebral artery occlusion using an intraluminal filament. The infarct volume, motor function, neurological deficits, and the cellular expressions levels of MK, BDNF, GFAP, PECAM-1, caspase 3, and nitrotyrosine (NT) were evaluated 48h after the induction of ischemia. The infarct volume, neurological deficits and motor function in the Ex group were significantly improved compared to that of the No-Ex group. The expression levels of MK, BDNF, GFAP, and PECAM-1 were enhanced in the Ex group compared to the expression levels in the No-Ex group after brain ischemia, while the expression levels of activated caspase 3 and NT were reduced in the area surrounding the necrotic lesion. Our findings suggest that preconditioning exercise reduced the infract volume and ameliorated motor function, enhanced expression levels of MK and BDNF, increased astrocyte proliferation, increased angiogenesis, and reduced neuronal apoptosis and oxidative stress.

Circadian Clock Genes Are Essential for Normal Adult Neurogenesis, Differentiation, and Fate Determination

Adult neurogenesis creates new neurons and glia from stem cells in the human brain throughout life. It is best understood in the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Circadian rhythms have been identified in the hippocampus, but the role of any endogenous circadian oscillator cells in hippocampal neurogenesis and their importance in learning or memory remains unclear. Any study of stem cell regulation by intrinsic circadian timing within the DG is complicated by modulation from circadian clocks elsewhere in the brain. To examine circadian oscillators in greater isolation, neurosphere cultures were prepared from the DG of two knockout mouse lines that lack a functional circadian clock and from mPer1::luc mice to identify circadian oscillations in gene expression. Circadian mPer1 gene activity rhythms were recorded in neurospheres maintained in a culture medium that induces neurogenesis but not in one that maintains the stem cell state. Although the differentiating neural stem progenitor cells of spheres were rhythmic, evidence of any mature neurons was extremely sparse. The circadian timing signal originated in undifferentiated cells within the neurosphere. This conclusion was supported by immunocytochemistry for mPER1 protein that was localized to the inner, more stem cell-like neurosphere core. To test for effects of the circadian clock on neurogenesis, media conditions were altered to induce neurospheres from BMAL1 knockout mice to differentiate. These cultures displayed unusually high differentiation into glia rather than neurons according to GFAP and NeuN expression, respectively, and very few BetaIII tubulin-positive, immature neurons were observed. The knockout neurospheres also displayed areas visibly devoid of cells and had overall higher cell death. Neurospheres from arrhythmic mice lacking two other core clock genes, Cry1 and Cry2, showed significantly reduced growth and increased astrocyte proliferation during differentiation, but they generated normal percentages of neuronal cells. Neuronal fate commitment therefore appears to be controlled through a non-clock function of BMAL1. This study provides insight into how cell autonomous circadian clocks and clock genes regulate adult neural stem cells with implications for treating neurodegenerative disorders and impaired brain functions by manipulating neurogenesis.

A novel compound, denosomin, ameliorates spinal cord injury via axonal growth associated with astrocyte‐secreted vimentin

In the spinal cord injury (SCI) axon regeneration is inhibited by the glial scar, which contains reactive astrocytes that secrete inhibitory chondroitin sulphate proteoglycan (CSPG). We previously reported that a novel compound, denosomin, promotes axonal growth under degenerative conditions in cultured cortical neurons. In this study, we investigated the effects of denosomin on functional recovery in SCI mice and elucidated the mechanism though which denosomin induces axonal growth in the injured spinal cord. Denosomin was administered p.o. for 7 or 14 days to contusion mice. Behavioural evaluations and immunohistochemistry were done. Primary cultured cortical neurons and astrocytes were treated with denosomin to investigate the mechanism of axonal growth facilitation. Denosomin improved hind limb motor dysfunction and axonal growth, especially in the 5-HT-positive tracts across the scar and increased the density of astrocytes. Denosomin increased astrocyte proliferation, inhibited astrocytic death and increased the expression and secretion of vimentin in cultured astrocytes. Furthermore, vimentin increased axonal outgrowth in cultured neurons, even in the presence of inhibitory CSPG. Denosomin increased the number of vimentin-expressing astrocytes inside glial scars of SCI mice, and 5-HT-positive axonal growth occurred in a vimentin-associated manner. Denosomin increased the ratio of astrocytes that secrete vimentin as an axonal growth facilitator, which, we propose enhances axonal growth beyond the glial scar and promotes functional recovery in SCI mice. This study is the first to demonstrate this novel role of vimentin in SCI and drug-mediated modification of the inhibitory property of reactive astrocytes.

Subchronic ketamine treatment leads to permanent changes in EEG, cognition and the astrocytic glutamate transporter EAAT2 in mice

Ketamine is an NMDA receptor antagonist with psychotomimetic, dissociative, amnestic and euphoric effects. When chronically abused, ketamine users display deficits in cognition and information processing, even following long-term abstinence from the drug. While animal studies have shown evidence of behavioral changes and cognitive deficits that mimic those seen in humans within the period immediately following subchronic ketamine, a few animal studies have assessed long-term changes following cessation of ketamine exposure. To this end, the present study assessed event related potentials (ERPs) and EEG oscillations in mice exposed to subchronic ketamine following a 6month period of abstinence from the drug. Ketamine-treated mice showed no change in P20, but did show marked reductions in amplitude of the later N40 and P80 components, consistent with previous studies of acute ketamine exposure. Additionally, ketamine-treated animals showed a significant reduction in stimulus evoked theta oscillations. To assess the functional significance of these changes, mice were also assessed on a series of behavioral and cognitive tests, including progressive ratio (motivation), extinction (behavioral flexibility) and win-shift radial maze (spatial memory). Subchronic ketamine produced marked disruptions in reversal learning and spatial memory. Analysis of brains from ketamine-treated mice failed to show evidence of neuronal degeneration as determined by NueN immunohistochemistry, but did show increased astrocyte proliferation and decreased expression of the glial specific glutamate transporter, GLT-1. These results strongly suggest: 1) that subchronic ketamine induces significant changes in brain function that long exceed exposure to the drug; 2) that ketamine exposure in mice induces lasting cognitive impairments closely resembling those observed in human ketamine abusers; 3) that ERP and EEG measures are highly sensitive to alterations in brain function associated with reduced cognitive function; and 4) that the brain changes induced by chronic ketamine treatment are suggestive of long-term adaptive or plastic, rather than degenerative, changes.

FOXO3a inhibits TNF‐α‐ and IL‐1β‐induced astrocyte proliferation:Implication for reactive astrogliosis

Reactive astrogliosis is one of the pathological hallmarks of neurodegenerative diseases. Inflammatory cytokines, such as TNF-α and IL-1β, have been shown to mediate the reactive astrogliosis in neurodegenerative diseases; however, the molecular mechanism remains unclear. In this study, we investigated the role of transcription factor FOXO3a on astrocyte proliferation, one primary aspect of severe reactive astrogliosis. Our results confirmed that TNF-α and IL-1β increased astrocyte proliferation, as determined by Ki67 and BrdU immunostaining. Furthermore, we found that cytokine-mediated astrocyte proliferation was accompanied by an increase of the phosphorylation and reduced nuclear expression of FOXO3a. Intracranial injection of TNF-α and IL-1β induced astrocyte proliferation and hypertrophy, which was associated with reduced nuclear expression of Foxo3a in astrocytes. To determine the function of FOXO3a in astrocyte proliferation, wild type FOXO3a was overexpressed with adenovirus, which subsequently upregulated p27Kip1 and Gadd45α, and significantly inhibited cytokine-induced astrocyte proliferation. In contrast, overexpression of dominant negative FOXO3a decreased p27Kip1, upregulated cyclin D1 and promoted astrocyte proliferation. Along the same line, astrocytes isolated from Foxo3a-null mice have higher proliferative potential. In response to intracranial injection of cytokines, Foxo3a-null mice manifested severe astrogliosis in vivo. In conclusion, FOXO3a is important in restraining astrocyte proliferation during proinflammatory cytokine stimulation and loss of function of FOXO3a may be responsible for the proliferation of astrocytes in the severe form of reactive astrogliosis. Understanding the key regulatory role of FOXO3a in reactive astrogliosis may provide a novel therapeutic target during neuroinflammation.

Connexin43 is involved in the effect of endothelin‐1 on astrocyte proliferation and glucose uptake

In previous studies, we showed that endothelin-1 increased astrocyte proliferation and glucose uptake. These effects were similar to those observed with other gap junction inhibitors, such as carbenoxolone (CBX). Because 24-h treatment with endothelin-1 or CBX downregulates the expression of connexin43, the main protein forming astrocytic gap junctions, which can also be involved in proliferation, in this study, we addressed the possible role of connexin43 in the effects of endothelin-1. To do so, connexin43 was silenced in astrocytes by siRNA. The knock down of connexin43 increased the rate of glucose uptake, characterized by the upregulation of GLUT-1 and type I hexokinase. Neither endothelin-1 nor CBX were able to further increase the rate of glucose uptake in connexin43-silenced astrocytes. In agreement, no effects of endothelin-1 and CBX on GLUT-1 and type I hexokinase were observed in connexin-43 silenced astrocytes or in astrocytes from connexin43 knock-out (KO) mice. Our previous studies suggested a close relationship between glucose uptake and astrocyte proliferation. Consistent with this, connexin43-silenced astrocytes exhibited an increase in Ki-67, a marker of proliferation. The effects of ET-1 on retinoblastoma phosphorylation on Ser780 and on the upregulation of cyclins D1 and D3 were affected by the levels of connexin43. In conclusion, our results indicate that connexin43 participates in the effects of endothelin-1 on glucose uptake and proliferation in astrocytes. Interestingly, although the rate of growth in connexin43 KO astrocytes has been reported to be reduced, we observed that an acute reduction in connexin43 by siRNA increased proliferation and glucose uptake.

Variations in astrocyte and fibroblast response due to biomaterial particulates in vitro

The possible involvement of orthopedic biomaterial particles such as cobalt-chrome alloy (Co-Cr), ultrahigh molecular weight polyethylene (UHMWPE), titanium alloy (Ti-6Al-4V), and polymethyl methacrylate (PMMA) in the formation of glial and meningeal scars was investigated using an in vitro system. Cell lines were used as models for astrocytes and meningeal fibroblasts. They were incubated with varying concentrations of particle suspensions, after which proliferative and cytotoxic responses were quantified using MTT assay and Live/Dead microscopy. It was determined that relative particulate toxicity (arranged in decreasing order) to astrocytes is Co-Cr > Ti-6Al-4V > PMMA > UHMWPE, and toxicity to fibroblasts is PMMA > Co-Cr > Ti-6Al-4V > UHMWPE. Cell death caused by PMMA was mainly due to necrosis, while the rest of the particles induced apoptosis. Low quantities of Co-Cr and Ti-6Al-4V stimulate increased astrocyte proliferation rate. However, only the cells treated with titanium alloy caused upregulated transcription of reactive astrocyte markers such as glial fibrillary acidic protein, vimentin, nestin, and type IV collagen, suggesting the potential of titanium alloy alone to trigger glial scarring. None of the biomaterials tested promoted proliferation in fibroblasts implying that biomaterial particles are not directly involved in meningeal scar development.

Tumor suppressor in lung cancer-1 (TSLC1) functions as a glioma tumor suppressor

Tumor suppressor in lung cancer-1 (TSLC1) loss is common in many human cancers, including meningioma. In this study, we demonstrate that TSLC1 protein and RNA expression is lost in 60% to 65% of high-grade gliomas, and that TSLC1 reintroduction into glioma cells results in growth suppression. Moreover, Tslc1 loss in mice results in increased astrocyte proliferation in vivo and in vitro. These data indicate that TSLC1 functions as a glioma tumor suppressor.

Increased astrocyte proliferation in rats after running exercise

The aim in this study was to investigate whether physical exercise could induce astroglial proliferation in the frontoparietal cortex and dorsolateral striatum where extensive angiogenesis had been found after exercise in previous studies. Adult male Sprague Dawley rats ( n = 48) were used in four experimental groups. Animals were exercised 30 min each day on a treadmill on which repetitive locomotor movement was required, for 0 ( n = 12), 3 ( n = 12) or 6 ( n = 12) weeks, as well as 3-week exercise plus 3-week rest ( n = 12). Brain tissues of the exercised and non-exercised rats were processed for glial fibrillary acidic protein (GFAP) immunocytochemistry ( n = 6 × 4) and Western blotting ( n = 6 × 4) to evaluate regional astrocyte proliferation in the frontoparietal cortex and dorsolateral striatum. By using GFAP immunocytochemistry and stereological methods, we compared the density of astrocytes in the animals with or without exercise. In comparison to non-exercised animals, a significant ( p < 0.01) increase in the number of astrocytes was observed in both cortex and striatum of rats exercised for 3 or 6 weeks. Our data also indicated that astrocytic density continued to increase up to 6 weeks either with an additional 3 weeks of exercise ( p < 0.01) or 3 weeks of rest ( p < 0.01). In addition, Western blotting analysis showed an obvious increase in GFAP protein from cortex and striatum of exercised animals. Astrocytosis after exercise, coupled with angiogenesis, is thought to provide strength to the neurovascular unit (a construct consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix). Strengthening of this unit by exercise may protect blood-brain-barrier function following brain injury, such as that occurring after stroke.

Disruption of the hyaluronan‐based extracellular matrix in spinal cord promotes astrocyte proliferation

Astrocyte proliferation is tightly controlled during development and in the adult nervous system. In the present study, we find that a high-molecular-weight (MW) form of the glycosaminoglycan hyaluronan (HA) is found in rat spinal cord tissue and becomes degraded soon after traumatic spinal cord injury. Newly synthesized HA accumulates in injured spinal cord as gliosis proceeds, such that high-MW HA becomes overabundant in the extracellular matrix surrounding glial scars after 1 month. Injection of hyaluronidase, which degrades HA, into normal spinal cord tissue results in increased numbers of glial fibrillary acidic protein (GFAP)-positive cells that also express the nuclear proliferation marker Ki-67, suggesting that HA degradation promotes astrocyte proliferation. In agreement with this observation, adding high- but not low-MW HA to proliferating astrocytes in vitro inhibits cell growth, while treating confluent, quiescent astrocyte cultures with hyaluronidase induces astrocyte proliferation. Collectively, these data indicate that high-MW HA maintains astrocytes in a state of quiescence, and that degradation of HA following CNS injury relieves growth inhibition, resulting in increased astrocyte proliferation.

Pten Loss Causes Hypertrophy and Increased Proliferation of Astrocytes In vivo

Somatic mutations of PTEN are found in many types of cancers including glioblastoma, the most malignant astrocytic tumor. PTEN mutation occurs in 25 to 40% of glioblastomas but is rarely observed in low-grade glial neoplasms. To determine the role of Pten in astrocytes and glial tumor formation, we inactivated Pten by a Cre-loxP approach with a GFAP-cre transgenic mouse that induced Cre-mediated recombination in astrocytes. Pten conditional knockout mice showed a striking progressive enlargement of the entire brain. Increased nuclear and soma size was observed in both astrocytes and neurons, which contributed in part to the increase in brain size. Pten-deficient astrocytes showed accelerated proliferation in vitro and aberrant ongoing proliferation in adult brains in vivo. In contrast, neurons lacking Pten did not show alterations in proliferation. This study shows cell-type dependent effects of Pten loss in the adult brain, including increased astrocyte proliferation that may render astroglial cells susceptible to neoplastic transformation or malignant progression.

Neurotoxic effects of polymorphonuclear granulocytes on hippocampal primary cultures.

Many neurological insults and neurodegenerative disorders are accompanied by an acute inflammatory reaction that can contribute to neuronal damage. This inflammation involves infiltration of bloodborne polymorphonuclear leukocytes (PMNs) into the injured brain area. The role of inflammation in brain injury, however, is controversial, because recent studies suggest that inflammation may actually be beneficial in the recovery from brain damage. Therefore, we investigated the effects of pathophysiologically relevant concentrations of PMNs in vitro on mixed hippocampal primary cultures. Rat PMNs and peripheral blood lymphocytes were isolated by density centrifugation and cocultured with hippocampal cells for 24-72 h plus or minus an excitotoxic insult (50 microM kainic acid) or 6-h oxygen glucose deprivation. Cell death was analyzed by immunocytochemistry, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, and neuron-specific [2,2'-azino-bis(ethylbenzothiazoline-6-sulfonic acid)] assay. After 3 days of coculture in the absence of insult, PMNs caused massive neuron loss and dramatic morphological changes in glial cells (astrocyte detachment, aggregation). Furthermore PMNs exacerbated kainic acid- and oxygen glucose deprivation-induced neuron death by 20-30%. The cytotoxic effect of PMNs required heterocellular contact and were ameliorated by protease inhibitors. Lymphocytes, on the other hand, were not neurotoxic, but, instead, increased astrocyte proliferation. These findings suggest that PMN might represent a harmful part of inflammation after brain injury that can contribute to secondary damage.

Role of the Rap1 GTPase in astrocyte growth regulation.

Tuberous sclerosis complex (TSC) is an autosomal dominant syndrome in which affected individuals develop nervous system abnormalities that might reflect astrocyte dysfunction. The TSC2 gene product, tuberin, encodes a GTPase-activating protein (GAP) domain, which regulates the activity of Rap1 in vitro. To determine whether dysregulated Rap1, resulting from TSC2 inactivation, leads to increased astrocyte proliferation in vivo, we generated transgenic mice expressing activated Rap1(G12V) specifically in astrocytes. We observed no statistically significant difference in the number of astrocytes between wild-type and GFAP-Rap1(G12V) littermates in vivo; however, during log-phase growth, we observed a 25% increase in GFAP-Rap1(G12V) astrocyte doubling times compared to wild-type controls. This decreased proliferation was associated with delayed MAP kinase, but not AKT, activation. Lastly, to determine whether constitutive Rap1 activation could reverse the increased astrocyte proliferation observed in transgenic mice expressing oncogenic Ras(G12V), we generated transgenic mice expressing both Ras(G12V) and Rap1(G12V) in astrocytes. These double transgenic mice showed a striking reversion of the Ras(G12V) astrocyte growth phenotype. Collectively, these results argue that the tumor suppressor properties of tuberin are unlikely to be related to Rap1 inactivation and that Rap1 inhibits mitogenic Ras pathway signaling in astrocytes.