The Expression of FBP1 after Traumatic Brain Injury and Its Role in Astrocyte Proliferation

Far upstream element binding protein 1 (FBP1) has been identified as an upstream gene of p27^kip1 (p27), which is a key regulator of mammalian cell cycle regulation and neurogenesis. To elucidate the expression and function of FBP1 in central nervous system lesion and repair, we performed a traumatic brain injury (TBI) model in adult rats. We observed that FBP1 protein level significantly reduced at day 3 after injury, and the downregulation of FBP1 was predominant in astrocytes, which were largely proliferated after injury. Furthermore, in vitro, overexpression of FBP1 was concomitant with the up-regulation of p27 and reduction of PCNA in LPS-induced astrocyte proliferation. These results suggest that a decreased level of FBP1 in brain is involved in the proliferation of glial cells after TBI.     

Leukemia inhibitory factor is a key regulator of astrocytic, microglial and neuronal responses in a low-dose pilocarpine injury model

Insult to the central nervous system (CNS) induces many changes, including altered neurotransmitter expression, activation of astrocytes and microglia, neurogenesis and cell death. Cytokines and growth factors are candidates to be involved in astrocyte and microglial activation, and the up-regulation of glial fibrillary acidic protein (GFAP) is associated with brain damage. One of these candidates is leukemia inhibitory factor (LIF), a pro-inflammatory cytokine that is induced in astrocytes by brain damage or seizure. LIF also regulates expression of both neuropeptide Y (NPY) and galanin following peripheral nerve injury. To test the hypothesis that LIF regulates astrocyte, microglial and neuropeptide responses to a mild insult, we used a low-dose pilocarpine model to induce a brief seizure in LIF knock-out (KO) mice. Compared to wild type mice, the LIF KO mouse displays reduced astrocyte and microglial activation in the hippocampus. In addition, LIF KO mice display dramatically altered NPY, but not galanin, expression in response to injury. Thus, LIF is required for normal glial responses to brain damage, and, as in the periphery, LIF regulates NPY expression in the CNS.     

Statins decrease chondroitin sulfate proteoglycan expression and acute astrocyte activation in central nervous system injury

Statins elicit numerous favorable effects on central nervous system (CNS) injury, including inhibition of the rhoA/ROCK pathway. In the present study, we show that statins decrease acute astrocyte activation in CNS injury, and decrease chondroitin sulfate proteoglycan (CSPG) levels in astrocyte cultures as well as CNS injury. CSPG levels decreased by up to 45% in simvastatin-treated astrocyte cultures compared to control cultures. In simvastatin-treated animals, CSPG levels declined by 60% 8 days after brain stab injury, and by 62-64% 4 weeks after spinal cord injury (SCI). Glial fibrillary acid protein (GFAP) levels decreased in brain stab at 8 days after surgery/intervention, suggesting that statins produce a decrease in astrocyte activation. Attenuation of astrocyte activation may contribute to the decline in CSPG levels. However, there are likely other contributing factors, since GFAP levels were not a contributing factor in the decline of CSPG levels in astrocyte cultures. Robust locomotor improvements were not observed with any treatment. The numerous beneficial effects of statins on CNS injury render them an attractive candidate in the treatment of CNS injury.     

SC1: a marker for astrocytes in the adult rodent brain is upregulated during reactive astrocytosis

Astrocytes are the most abundant cell type in the mammalian central nervous system (CNS), and are involved in many processes critical for normal CNS maintenance and function. We have used double-label immunocytochemistry and in situ analysis to show that the SPARC (secreted protein acidic and rich in cysteine)-related protein SC1, co-localizes with the astrocyte marker glial fibrillary acidic protein (GFAP) in the adult rodent brain. Thus, SC1 is an astrocyte marker that may be used to investigate astrocyte heterogeneity and analyze glial cell lineages during neural development. Consistent with the presence of SC1 and GFAP in astrocytes, both proteins were markedly upregulated following reactive astrocytosis induced by focal mechanical trauma. Therefore, SC1 may play an important role in reactive astrocytosis subsequent to a wide variety of neural trauma, including neurodegenerative diseases and acute neural damage.     

TNF alpha and IL-1beta mediate intercellular adhesion molecule-1 induction via microglia-astrocyte interaction in CNS radiation injury

Radiation injury to the central nervous system (CNS) results in glial activation accompanied by expression of pro-inflammatory cytokines and adhesion molecules. In this study we demonstrate intercellular adhesion molecule-1 (ICAM-1) induction in the irradiated mouse brain at the mRNA and protein levels. Immunocytochemical analysis revealed that ICAM-1 protein was primarily expressed in endothelial cells and microglia. In vitro, ionizing radiation significantly induces TNF alpha, IL-1beta and ICAM-1 mRNA in primary microglia cultures. Interestingly, although ionizing radiation activated primary astrocyte cultures, it did not induce ICAM-1 expression. However, exposure of astrocytes to conditioned medium collected from irradiated microglia resulted in ICAM-1 induction, which was abrogated when the conditioned medium was pre-incubated with neutralizing antibodies raised against murine TNF alpha and IL-1beta. These results indicate that pro-inflammatory cytokines may be necessary for ICAM-1 expression in astrocytes in CNS radiation injury.     

Traumatic Brain Injury Induces a Downregulation of MSK1 in Rat Brain Cortex

Mitogen- and stress-activated protein kinase (MSK) 1 protein was initially identified as a particularly interesting protein of mitogen-activated protein kinase. It was reported to enhance B cell lymphoma 2-associated death protein’s phosphorylation to protect cell death, suggesting that MSK1 represents a new type of anti-cell death gene. Moreover, a recent study has shown that MSK1 is involved in negative feedback pathways that are crucial to prevent uncontrolled inflammation. However, its function and expression in the central nervous system lesion are not been understood very well. In this study, we performed a traumatic brain injury (TBI) model in adult rats and investigated the dynamic changes of MSK1 expression in the brain cortex. Double immunofluorescence staining revealed that MSK1 was co-expressed with neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP). Besides, co-localization of MSK1/active caspase 3 and MSK1/proliferating cell nuclear antigen (PCNA) was detected in NeuN and GFAP. We also examined the expression profiles of PCNA and active caspase 3 whose changes were correlated with the expression of MSK1. All our findings suggested that MSK1 might be involved in the pathophysiology of brain after TBI.     

Osteopontin is indispensable for activation of astrocytes in injured mouse brain and primary culture

ABSTRACT

Objectives: Osteopontin (OPN) is an inflammatory cytokine inducer involved in cell proliferation and migration in inflammatory diseases or tumors. To investigate the function of OPN in astrocyte activation during brain injury, we compared OPN-deficient (OPN/KO) with wild-type (WT) mouse brains after stab wound injury and primary culture of astrocytes.

Methods: Primary cultures of astrocytes were prepared from either WT or OPN/KO postnatal mouse brains. Activation efficiency of astrocytes in primary culture was accessed using Western blotting by examining the protein levels of glial fibrillary acidic protein (GFAP) and tenascin-C (TN-C), which are markers for reactive astrocytes, following lipopolysaccharide (LPS) stimulation. Furthermore, the stab wound injury on the cerebral cortex as a brain traumatic injury model was used, and activation of astrocytes and microglial cells was investigated using immunofluorescent analysis on fixed brain sections.

Results: Primary cultures of astrocytes prepared from WT or OPN/KO postnatal mouse brains showed that only 25% of normal shaped astrocytes in a flask were produced in OPN/KO mice. The expression levels of both GFAP and TN-C were downregulated in the primary culture of astrocytes from OPN/KO mice compared with that from WT mice. By the immunofluorescent analysis on the injured brain sections, glial activation was attenuated in OPN/KO mice compared with WT mice.

Discussion: Our data suggest that OPN is essential for proper astrocytic generation in vitro culture prepared from mouse cerebral cortex. OPN is indispensable for astrocyte activation in the mouse brain injury model and in LPS stimulated primary culture.

Abbreviations: AQP4: aquaporin 4; BBB: blood brain barrier; BrdU: bromo-deoxy uridine; CNS: central nervous system; GFAP: glial fibllirary acidic protein; IgG: immunoglobulin G; LPS: lipopolysaccharide; OPN: osteopontin; OPN/KO: osteopontin-deficient; TN-C: tenascin-C     

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.     

Bone morphogenetic proteins mediate cellular response and, together with Noggin, regulate astrocyte differentiation after spinal cord injury

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.     

Assessment of neurotoxicity of monocrotaline, an alkaloid extracted from Crotalaria retusa in astrocyte/neuron co-culture system

Studies have shown cases of poisoning with plants from the genus Crotalaria (Leguminosae) mainly in animals. They induce damages in the central nervous system (CNS), which has been attributed to toxic effects of the pyrrolizidine alkaloid (PA) monocrotaline (MCT). Previously we demonstrated that both MCT and dehydromonocrotaline (DHMC), its main active metabolite, induce changes in the levels and patterns of expression of the main protein from astrocyte cytoskeleton, glial fibrillary acidic protein (GFAP). In this study we investigated the effect of MCT on rat cortical astrocyte/neuron primary co-cultures. Primary cultures were exposed to 10 or 100 μM MCT. The MTT test and the measurement of LDH activity on the culture medium revealed that after 24h exposure MCT was not cytotoxic to neuron/astrocyte cells. However, the cell viability after 72 h treatment decreased in 10-20%, and the LDH levels in the culture medium increased at a rate of 12% and 23%, in cultures exposed to 10 or 100 μM MCT. Rosenfeld staining showed vacuolization and increase in cell body in astrocytes after MCT exposure. Immunocytochemistry and Western blot analyses revealed changes on pattern of GFAP and βIII-tubulin expression and steady state levels after MCT treatment, with a dose and time dependent intense down regulation and depolarization of neuronal βIII-tubulin. Moreover, treatment with 100 μM MCT for 12h induced GSH depletion, which was not seen when cytochrome P450 enzyme system was inhibited indicating that it is involved in MCT induced cytotoxicity in CNS cells.     

Astrocyte proliferation in the chick auditory brainstem following cochlea removal

Astrocytes in the central nervous system (CNS) respond to injury and disease by proliferating and extending processes. The intermediate filament protein of astrocytes, glial fibrillary acidic protein (GFAP) also increases in astrocytes. These cells are called “reactive astrocytes” and are thought to play a role in CNS repair. We have previously demonstrated rapid increases (< 6 hours) in GFAP-immunoreactive and silver-impregnated glial processes in the chick cochlear nucleus, nucleus magnocellularis (NM), following cochlea removal or activity blockade of the eighth nerve. It was not known whether these changes were the result of glial proliferation, glial hypertrophy, or both. The present study examined the time course of astrocyte proliferation in NM following cochlea removal. Postnatal chicks received unilateral cochlea removal and survived for 6, 12, 18, 24, 36, 48, and 72 hours. Bromodeoxyuridine was used to label proliferating cells. The volume and number of labeled cells in NM was calculated for both the experimental and control sides of the brains for experimental animals was well as for unoperated control animals. A subset of astrocytes continuously divide in the normal posthatch chick brainstem. The percentage of labeled nuclei increases within NM 36 hours following cochlea removal and is robust by 48 hours. This increase is due to astrocyte proliferation within, rather than migration to, NM. These resulis indicate that rapid increases in GFAP following reduced activity are independent of cell proliferation. The time course of astrocyte proliferation suggests that cellular degeneration within the nucleus may play a role in upregulating astrocyte proliferation. © 1994 Wiley-Liss, Inc.     

In the hypoxic central nervous system,endothelial cell proliferation is followed by astrocyte activation,proliferation, and increased expression of the α6β4 integrin and dystroglycan

Cerebral hypoxia induces a profound angiogenic response in the central nervous system (CNS). Using a mouse model of chronic cerebral hypoxia, we previously demonstrated that angiogenic vessels in the hypoxic CNS show marked upregulation of the extracellular matrix (ECM) protein fibronectin, along with increased expression of its major receptor, α5β1 integrin on brain endothelial cells (BEC). As cerebral hypoxia also leads to glial activation, the aim of the current study was to define the temporal relationship between BEC responses and glial cell activation in this model of cerebral hypoxia. This revealed that BEC fibronectin/α5β1 integrin expression and proliferation both reached maximal level after 4‐day hypoxia. Interestingly, up to 4‐day hypoxia, all dividing cells were BEC, but at later time‐points proliferating astrocytes were also observed. GFAP staining revealed that hypoxia induced marked astrocyte activation that reached maximal level between 7‐ and 14‐day hypoxia. As newly formed cerebral capillaries require ensheathment by astrocyte end‐feet to acquire mature brain endothelium characteristics, we next examined how expression of astrocyte end‐feet adhesion molecules is regulated by hypoxia. This showed that the astrocyte adhesion receptors α6β4 integrin and dystroglycan were both markedly upregulated, with a time‐course that closely resembled astrocyte activation. Taken together, this evidence shows that cerebral hypoxia promotes first an endothelial response, in which fibronectin promotes BEC proliferation. This is then followed by an astrocyte response, involving astrocyte activation, proliferation, and reorganization of astrocyte end‐feet, which correlates with increased expression of astrocyte end‐feet adhesion molecules. © 2010 Wiley‐Liss, Inc.     

Tyrosine phosphatase SHP-1 immunoreactivity increases in a subset of astrocytes following deafferentation of the chicken auditory brainstem

Proliferation of astrocytes is a dramatic response of the central nervous system (CNS) to injury and disease. Such proliferation results in the formation of the neural/glial scar and the reconstitution of the glial limitans. However, not all astrocytes enter the proliferative cycle following injury, and for those that do, the period of cell division is limited. Little attention has focused on the events that regulate the duration and extent of astrocyte proliferation following damage, but clearly control mechanisms are in place as CNS injury does not result in the continuous astrocyte proliferation seen in glial tumorigenesis. Protein tyrosine phosphorylation has been implicated in both astrocyte proliferation and differentiation and plays an important role in the regulation of the cell cycle in a number of different systems. We have found a small subset of astrocytes in the chick auditory brainstem that are immunopositive for the protein tyrosine phosphatase SHP-1. SHP-1 appears to negatively regulate cellular division in the hematopoietic system and is involved in the mitogenic response to various growth factors. Following cochlea removal, there is a marked increase within the auditory brainstem nucleus, nucleus magnocellularis (NM), in both in the number of SHP-1-positive astrocytes and the length of their immunopositive fibers. Significantly, those animals showing the greatest increases in SHP-1 immunoreactivity do not exhibit large amounts of astrocyte proliferation. We hypothesize that the expression of SHP-1 plays a role in negatively regulating the mitotic behavior of astrocytes following deafferentation.     

Inhibition of reactive astrocytosis in established experimental autoimmune encephalomyelitis favors infiltration by myeloid cells over T cells and enhances severity of disease

Reactive astrocytosis, involving activation, hypertrophy, and proliferation of astrocytes, is a characteristic response to inflammation or injury of the central nervous system. We have investigated whether inhibition of reactive astrocytosis influences established experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We made use of transgenic mice, which express herpes simplex virus-derived thymidine kinase under control of a glial fibrillary acidic protein promotor (GFAP HSV-TK mice). Treatment of these mice with ganciclovir leads to inhibition of reactive astrocytosis. When GFAP HSV-TK mice were treated for seven days following onset of EAE with ganciclovir, disease severity increased. Although aquaporin-4 staining on astrocyte endfeet at the glia limitans remained equally detectable, GFAP immunoreactivity and mRNA expression in CNS were reduced by this treatment. Ganciclovir-treated GFAP HSV-TK mice with EAE had a 78% increase in the total number of infiltrating myeloid cells (mainly macrophages), whereas we did not find an increase in infiltrating T cells, using quantitative flow cytometry. Per cell expression of mRNA for the macrophage-associated molecules TNFα, MMP-12 and TIMP-1 was elevated in spinal cord of GFAP HSV-TK mice treated with ganciclovir. Relative expression of CD3ε was downregulated, and expression levels of IFNγ, IL-4, IL-10, IL-17, and Foxp3 were not significantly changed. mRNA expression of CCL2 was upregulated, and CXL10 was downregulated. Thus, inhibition of reactive astrocytosis after initiation of EAE leads to increased macrophage, but not T cell, infiltration, and enhanced severity of EAE. This emphasizes the role of astrocytes in controlling leukocyte infiltration in neuroinflammation.     

Isolation of cDNA clones encoding rat glial fibrillary acidic protein: expression in astrocytes and in Schwann cells.

Glial fibrillary acidic protein (GFAP) expressed by astrocytes in the central nervous system (CNS) has been extensively characterized but the molecular identity of related molecules in the peripheral nervous system (PNS) remains unclear. To examine possible structural differences between CNS and PNS GFAP, we have isolated cDNA clones for rat GFAP from both cultured astrocyte and Schwann cell libraries. Nucleotide sequence analysis indicated that the PNS and CNS GFAP clones contained identical coding regions, with a predicted protein product of 430 amino acids. However, the 5'-untranslated region of clone rGFA15, isolated from the Schwann cell library, was longer than that predicted for brain-derived GFAP mRNA. Primer extension analysis of RNA isolated from the RT4-D6 Schwann cell line indicated that the start site for PNS GFAP mRNA lies 169 bases upstream from that used in the CNS. In addition, tryptic peptide mapping of GFAP prepared from cultured astrocytes and Schwann cells revealed one major peptide fragment present in CNS GFAP but absent from PNS GFAP. These results suggest structural differences between GFAP in these two cell types, at both the nucleic acid and protein level, and are consistent with previous observations of immunochemical differences existing between CNS and PNS GFAP.     

Reactive astrocyte formation in vivo is regulated by noradrenergic axons

Beta adrenergic receptor antagonists greatly reduce reactive astrocyte formation induced by neuronal degeneration. To test the hypothesis that the density of noradrenergic innervation is a factor in the regulation of astrocytosis, we measured glial fibrillary acidic protein (GFAP) optical density after neuronal injury in central nervous system (CNS) regions with permanent noradrenergic sprouting or norepinephrine (NE) depletion. The injury model employs the injection of Ricinus communis lectin into a cranial or peripheral nerve to destroy CNS neurons without the blood-brain barrier disruption and lymphocyte infiltration associated with contusive or surgical lesions. We took advantage of the lack of an NE transporter in the terminals of certain classes of noradrenergic axons to produce noradrenergic sprouting in the trigeminal motor nucleus (MoV) with neonatal 6-hydroxydopamine (6-OHDA) treatment and to produce depletion of NE in the spinal cord dorsal horn with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4) administration. In each of these regions, GFAP optical density in the region of reactive astrocytes on the Ricin lectin-treated side was compared with the untreated contralateral (control) side in animals with NE hyperinnervation or NE depletion. GFAP density was increased about 55% in the injured NE-hyperinnervated MoV and was decreased about 35% in the injured NE-depleted dorsal horn. The degree of reactive astrocyte formation to injury is known to vary in different regions of the CNS, and our results suggest that differences in noradrenergic innervation may contribute to this variation. Along with earlier findings that β-adrenergic receptor blockade reduces reactive astrocyte formation, these data indicate that the noradrenergic innervation is a factor in the degree of astrocyte reactivity following injury. © 1996 Wiley-Liss, Inc.