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Spinal cord injury produced by mechanical contusion causes the onset of acute and chronic degradative events. These include blood brain barrier disruption, edema, demyelination, axonal damage and neuronal cell death. Posttraumatic inflammation after spinal cord injury has been implicated in the secondary injury that ultimately leads to neurologic dysfunction. Studies after spinal cord contusion have shown expression of several chemokines early after injury and suggested a role for them in the ordered recruitment of inflammatory cells at the lesion site (McTigue et al. [1998] J. Neurosci. Res. 53:368-376; Lee et al., [2000] Neurochem Int). We have demonstrated previously that infusion of the broad-spectrum chemokine receptor antagonist (vMIPII) in the contused spinal cord initially attenuates leukocyte infiltration, suppresses' gliotic reaction and reduces neuronal damage after injury. These changes are accompanied by increased expression of bcl-2, the endogenous apoptosis inhibitor, and reduced neuronal apoptosis (Ghirnikar et al. [2000] J. Neurosci. Res. 59:63-73). We demonstrate that 2 and 4 weeks of vMIPII infusion in the contusion-injured spinal cord also results in decreased hematogenous infiltration and is accompanied by reduced axonal degeneration in the gray matter. Luxol fast blue and MBP immunoreactivity indicated reduced myelin breakdown in the dorsal and ventral funiculi. Increased neuronal survival in the ventral horns of vMIPII infused cords was seen along with increased bcl-2 staining in them. Immunohistochemical identification of fiber phenotypes showed increased presence of calcitonin gene related peptide, choline acetyl transferase and tyrosine hydroxylase positive fibers as well as increased GAP43 staining in treated cords. These results suggest that sustained reduction in posttraumatic cellular infiltration is beneficial for tissue survival. A preliminary report of this study has been published (Eng et al. [2000] J. Neurochem. 74(Suppl):S67B). In contrast to vMIPII, infusion of MCP-1 (9-76), a N-terminal analog of the MCP-1 chemokine showed only a modest reduction in cellular infiltration at 14 and 21 dpi without significant tissue survival after spinal cord contusion injury. Comparing data on tissue survival obtained with vMIPII and MCP-1 (9-76) further validate the importance of the use of broad-spectrum antagonists in the treatment of spinal cord injury. Controlling the inflammatory reaction and providing a growth permissive environment would enhance regeneration and ultimately lead to neurological recovery after spinal cord injury. J. Neurosci. Res. 64:582-589, 2001. Published 2001 Wiley-Liss, Inc.

Spinal cord injury is accompanied by an initial inflammatory reaction followed by secondary injury that is caused, in part, by apoptosis. Recruitment of leukocytes from the blood compartment to the site of inflammation in the injured spinal cord has been attributed to locally generated chemotactic agents (cytokines and chemokines). In addition to upregulation in the message levels of a number of chemokines, we have found up-regulation in the message levels of several chemokine receptors following spinal cord contusion injury. To reduce the inflammatory response after spinal cord injury, we have blocked the interaction of chemokine receptors with their ligands using the vMIPII chemokine antagonist. Using a rat model of spinal cord contusion injury, we show that continuous infusion of the antagonist for up to 7 days results in a decrease in infiltrating hematogenous cells at the site of injury. Histological evaluation ofthe tissue showed fewer activated macrophages at the site of injury. Concomitantly, reduced neuronal loss and gliosis were observed in the antagonist infused spinal cord. In addition, increased expression of Bcl-2 gene, an endogenous inhibitor of apoptosis, was seen in the antagonist infused spinal cord at 7 days post injury. Morphologically, staining with the bisbenzamide dye Hoechst 33342 showed significantly more apoptotic bodies in the vehicle compared to antagonist infused spinal cord. Our data suggest that chemokine antagonist infusion post-injury results in limiting the inflammatory response following spinal cord contusion injury, thereby attenuating neuronal loss, possibly due to decreased apoptosis. These findings support the contention that disrupting chemokine interactions with their receptors may be an effective approach in reducing the secondary damage after spinal cord injury.

The present study evaluates the physiological effects of magnetic stimulation on astrocyte cultures. Cell cultures were exposed to pulsed magnetic stimulation (10 Hz, 10 sec) at the following levels: 0.10 tesla (T; Group A); 0.21 T (Group B); 0.42 T (Group C); and 0.63 T (Group D). Glial fibrillary acidic protein (GFAP) levels from immunoblots, total protein concentrations, and cellular morphology were analyzed at 0, 1, 3, 5, 7, 13, and 20 days poststimulation. Significantly higher GFAP levels were observed in Group D at day 3 (P = 0.0114). The change was transient as the GFAP levels quickly returned to control levels by day 5. No other significant changes in GFAP levels were observed. In comparison to control protein levels at day 0, concentrations from Groups B, C, and D were significantly lower (P < 0.006), whereas at day 3, Groups C and D were significantly higher (P < 0.02). Differences in astrocyte morphology due to magnetic stimulation were not observed. This study demonstrated that high intensity magnetic stimulation for only 10 sec induced a transient biological response.

Mice carrying copies of the human glial fibrillary acidic protein (hGFAP) gene driven by its own promoter have been generated that express the human transgene at different levels (Messing et al.: 152:391-398, 1998). Lines that expressed high levels of the gene died shortly after birth. Astrocyte cultures prepared from a low overexpressor (Tg73.2) exhibited abnormal cytoplasmic inclusions identical to those seen in vivo in the high overexpressors. Astrocytes in the Tg73.2 cultures appear odd-shaped and enlarged, express increased levels of GFAP (both human and mouse), and express alphaB crystallin protein, Hsp27 protein, and vimentin protein. At the light microscopic level, the Tg73.2 astrocytes are filled with eosinophilic deposits surrounded by positive GFAP immunostain. Ultrastructurally, the Tg73.2 astrocytes contain osmophilic deposits on a bed of intermediate filaments identical to Rosenthal fibers found in the brain in Alexander's disease. It seems that Tg73.2 mouse astrocytes in culture do not require additional stress from external sources or contact with other neuroectodermal cells to produce Rosenthal fibers. This suggests that the added hGFAP gene is sufficient to induce Rosenthal fibers and that an excess of GFAP in astrocytes may be detrimental to normal function. We hypothesize that the normal mechanism for GFAP turnover may be insufficient to handle the excess GFAP, thus causing an accumulation of stress proteins. The increased amounts of stress proteins and GFAP results in the formation of Rosenthal fibers, similar to those found in Alexander's disease.

A traumatic injury to the adult mammalian central nervous system (CNS) results in reactive astrogliosis and the migration of hematogenous cells into the damaged neural tissue. Chemokines, a novel class of chemoattractant cytokines, are now being recognized as mediators of the inflammatory changes that occur following injury. The expression of MCP-1 (macrophage chemotactic peptide-1), a member of the beta family of chemokines, has recently been demonstrated in trauma in the rat brain (Berman et al.: J Immunol 156:3017-3023, 1996). Using a stab wound model for mechanical injury, we studied the expression of two other beta chemokines: RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) and MIP-1 beta (macrophage inflammatory protein-1 beta) in the rat brain. The stab wound injury was characterized by widespread gliosis and infiltration of hematogenous cells. Immunohistochemical staining revealed the presence of RANTES and MIP-1 beta in the injured brain. RANTES and MIP-1 beta were both diffusely expressed in the necrotic tissue and were detected as early as 1 day post-injury (dpi). Double-labeling studies showed that MIP-1 beta, but not RANTES, was expressed by reactive astrocytes near the lesion site. In addition, MIP-1 beta staining was also detected on macrophages at the site of injury. The initial expression of the chemokines closely correlated with the appearance of inflammatory cells in the injured CNS, suggesting that RANTES and MIP-1 beta may play a role in the inflammatory events of traumatic brain injury. This study also demonstrates for the first time MIP-1 beta expression in reactive astrocytes following trauma to the rat CNS.

Astrocytomas are the most common brain tumors arising in the CNS and account for 65% of all primary brain tumors. Astrocytes have been shown to have the highest predisposition to malignant transformation compared to any other CNS cell type. The majority of astrocytomas are histologically malignant neoplasm. Previous studies have shown that resident astrocytes are the first cell type to react to tumors and surround them. However, the role of these astrocytes in tumor formation and progression has not been determined. In the present study, we have co-cultured astrocytes with a permanent cell line S635c15 (derived from anaplastic astrocytoma) in order to understand the cellular interactions between astrocytes and astrocytoma cells. Our studies demonstrate that astrocytes in contact with the tumor cells become reactive and fibrous with an increase in glial fibrillary acidic protein (GFAP) immunoreactivity as early as 4 days in culture. By 8 days, astrocytes formed glial boundaries around the tumor cells which grew as round colonies. The astrocytic processes surrounding the tumor cells were also intensely GFAP positive. Since the behavior of these cells observed in culture is very similar to their interaction seen in vivo, this co-culture system may serve as an in vitro model for astrocyte and astrocytoma cell line interaction and aid in our understanding of the molecular and cellular mechanisms during early stages of tumor formation and cell interactions.

Macrophage inflammatory protein 1 (MIP-1) is a recently characterized inflammatory and chemokinetic cytokine. Proinflammatory stimuli have been shown to induce expression of MIP-1 by macrophages. We hypothesized that microglia and astrocytes express MIP-1 alpha because of their many immunologic similarities to macrophages. MIP-1 alpha mRNA was examined with quantitative reverse transcription and polymerase chain reaction in an immortalized mouse microglial cell line (BV-2) and in mouse cortical astrocyte cultures. We found that in both the BV-2 microglial cell line and in astrocyte cultures, MIP-1 alpha mRNA was strongly induced by lipopolysaccharide and the phorbol ester PMA. MIP-1 alpha mRNA was reduced by dBcAMP, interferon-gamma, and PGE1. Dexamethasone decreased MIP-1 alpha mRNA levels in astrocyte cultures, but not in BV-2 microglial cells. Interleukin-1 beta, tumor necrosis factor alpha, and MIP-1 alpha had no effect on MIP-1 alpha mRNA expression. These findings demonstrate that MIP-1 alpha mRNA is expressed by cultured glial cells and is regulated by proinflammatory and anti-inflammatory stimuli. MIP-1 alpha may be expressed by microglia and astrocytes in vivo, and may help modulate cerebral inflammation.

Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 micrograms/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis.

Injury to the central nervous system (CNS) either from trauma or due to demyelinating/degenerating diseases results in a typical response of astrocytes, termed astrogliosis. This reaction is characterized by astrocyte proliferation, extensive hypertrophy of nuclei, cell body, and cytoplasmic processes and an increase in immunodetectable glial fibrillary acidic protein (GFAP). GFAP accumulation may cause a physical barrier preventing the reestablishment of a functional environment. Our studies have aimed at modulating astrogliosis by inhibiting or delaying GFAP synthesis in damaged and reactive astrocytes. The present study investigates the use of a recombinant retrovirus expressing antisense GFAP RNA in controlling the response of mechanically injured astrocytes. A 650 bp fragment from the coding region of mouse GFAP cDNA was cloned in the antisense orientation under the control of long terminal repeat (LTR) promoter of Moloney murine leukemia virus. Increase in GFAP as detected by immunocytochemical staining in injured astrocytes was inhibited by treatment with retrovirus expressing antisense GFAP RNA. Also, astrocytes at the site of injury in these scratched cultures did not show cell body hypertrophy compared to control cultures. These observations demonstrate that the increase in GFAP at the site of injury can be inhibited using retroviral treatment and indicate the potential of retrovirus-mediated gene transfer in modulating scar formation in the CNS in vivo. These studies also shed light on the role of GFAP in maintaining the morphology of astrocytes.

The reverse transcription and polymerase chain reaction technique (RT-PCR) was assessed for the quantification of changes in mRNA levels from primary astrocyte cultures. The effects of dibutyryl cyclic AMP (dBcAMP) on glial fibrillary acidic protein (GFAP) mRNA and the effects of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), and lipopolysaccharide (LPS) on interleukin-6 (IL-6) mRNA were examined. Two quantitative PCR methods were used: one involved carrying out the reaction in the exponential phase and the other involved the coamplification of a competitive target sequence. Increased GFAP mRNA in response to chronic dBcAMP treatment and increased IL-6 mRNA in response to TNF-alpha/IL-1 beta were readily detected. Both RT-PCR techniques were found to be suitable for the detection of large as well as smaller (twofold) changes in mRNA levels. The advantages and limitations of RT-PCR for mRNA quantification are discussed.

Astrogliosis is a predictable response of astrocytes to various types of injury caused by physical, chemical, and pathological trauma. It is characterized by hyperplasia, hypertrophy, and an increase in immunodetectable glial fibrillary acidic protein (GFAP). As GFAP accumulation is one of the prominent features of astrogliosis, inhibition or delay in GFAP synthesis in damaged and reactive astrocytes might affect astrogliosis and delay scar formation. The aim of this study is to investigate the possibility of utilizing antisense oligonucleotides in controlling the response of astrocytes after mechanically induced injury. We scratched primary astrocyte cultures prepared from newborn rat cerebral cortex with a plastic pipette tip as an injury model and studied the astrogliotic responses in culture. Injured astrocytes became hyperplastic, hypertrophic, and had an increased GFAP content. These observations demonstrate that injured astrocytes in culture are capable of becoming reactive and exhibit gliotic behaviors in culture without neurons. The increase in GFAP content in injured astrocytes could be inhibited by incubating the scratched culture with commercially available liposome complexed with 3' or 5' antisense oligonucleotides (20 nt) in the coding region of mouse GFAP. The scratch model provides a simple system to examine in more detail the mechanisms involved in triggering glial reactivity and many of the cellular dynamics associated with scar formation. Antisense oligonucleotide treatment could inhibit the GFAP synthesis in injured astrocytes, hence it may be applicable in modifying scar formation in CNS injury in vivo.

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with Lipofectin (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7-8.8 eta g per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 eta g per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30-40% decrease in GFAP content 2 days after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial fibrillary acidic protein (GFAP) is the major constituent of glial filaments and is restricted within the CNS to astrocytes. As with other classes of intermediate filament proteins, the regulation of GFAP expression is poorly understood. Utilizing highly purified cultures of astrocytes and a chemically defined (CD) medium, we have demonstrated that the expression of GFAP is subject to regulation by hormones and growth factors. The concentration of GFAP/mg protein was induced 2-4-fold in the presence of hydrocortisone, putrescine, prostaglandin F-2 alpha (PGF2 alpha), and pituitary fibroblast growth factor (FGF). Augmentation of the levels of GFAP continued for up to 3 weeks after conversion to CD medium and paralleled the morphological maturation of astrocytes. The accumulation of GFAP resulted from an increase in its specific rate of synthesis. Conversion of astrocytes from serum-supplemented (SS) to CD medium did not alter its rate of degradation. GFAP appeared quite stable under both sets of conditions, exhibiting a half-life of approximately 7.5 days. The data demonstrate that GFAP expression in astrocytes is subject to hormonal regulation, which may have implications for gliosis.

Neonatal rat brain astrocyte secondary cultures were established on nitrocellulose membrane filters (13-mm diameter Millipore disk) and on plastic coverslips in serum-supplemented medium. On these substrata, cultured astrocytes changed their shape from flat and polygonal to stellate in the absence of hormones or growth factor supplements. Cultures became confluent after 4 days, and astrocytes on nitrocellulose filters continued to differentiate morphologically and biochemically, as evidenced by extensive cytoplasmic process formation and glial fibrillary acid protein (GFAP) accumulation. Cultures were immunostained for GFAP and vimentin. mRNAs to GFAP, vimentin, alpha and beta tubulin, and actin also were detected by in situ hybridization with biotinylated cDNA probes. The astrocyte culture method on nitrocellulose provides a simple, versatile means of comparing undifferentiated, morphologically mature, reactive, and neoplastic astrocytes in vitro.

The gliotic scar in the demyelinated plaque is a prominent feature of the pathology of multiple sclerosis. Chronic relapsing experimental allergic encephalomyelitis (EAE) in the SJL/J mouse has many characteristics in common with multiple sclerosis in the human, including the development of intense gliosis during the course of the demyelinating disease. With the use of antibody to the astrocyte marker glial fibrillary acidic protein (GFAP) we have measured the increase of GFAP over an 11-month course of chronic EAE. Intense staining of astrocyte fibers was seen around EAE lesions, which were most frequently observed in the cerebellum, periventricular areas, and in the spinal cord. The relative amount of GFAP was estimated by preparation of cytoskeletal proteins from the affected CNS areas, separation of proteins by polyacrylamide gel electrophoresis, and quantitation of GFAP in relation to the 70-kD neurofilament protein (NF) in gel scans. The ratio GFAP/70-kD NF protein in control animals did not change significantly over 11 months, whereas this ratio gradually increased to 2.54 in animals with chronic relapsing EAE 6 months after immunization. Although some decrease of neural fibers may have contributed partially to this change in ratio, the amounts of GFAP were greatly increased. These results indicate that the SJL/J mouse with chronic relapsing EAE provides an excellent model with which to investigate the formation and development of the gliotic plaque analogous to that seen in demyelinated areas in multiple sclerosis tissue.