Emeritus Faculty, Acad Council, Pathology
Astrocytes make up a substantial proportion of the central nervous system (CNS) and participate in a variety of important physiologic and pathologic processes. They are characterized by vigorous response to diverse neurologic insults and induction of the glial fibrillary acidic protein (GFAP) gene, a feature that is well conserved across a variety of different species. The prominence of astroglial reactions in various diseases, the rapidity of the astroglial response and the evolutionary conservation of reactive astrogliosis indicate that reactive astrocytes fulfill important functions of the CNS. It is important to identify the essential molecular mechanisms which activate metabolic responses in astrocytes in order to understand astrogliosis. Our experimental models include:
1) Primary dissociated rodent brain astrocyte cultures,
2) Growth factor transfected astrocytes transplanted into contused rat spinal cord,
3) Brain stab wound. Longitudinal studies are being carried out to determine the induction of cytoskeletal proteins, cytokines, early response genes, and growth factors in the mechanical injury model of rat spinal cord and in a stab wound injury to mouse and rat brains.
The study employs quantitative reverse transcriptase-polymerase chain reaction, immunocytochemistry, in situ hybridization, ELISA assays, Western blot, and Northern blot. Normal and transgenic mice containing a transgenic vector composed of a 2.2 kb 5'-flanking sequence derived from the GFAP gene fused to the E coli lacZ structural gene GFAP null, and GFAP overexpressing mice are being used. Some of the substances to be determined are c-Fos, c-Jun, heat shock protein, IL-1, IL-6, TNFa, NGFb, CNTF, EGF, PDGF, GFAP, LIF, MIP, vimentin, and actin. Identification of those factors that promote and those that inhibit regeneration will allow one to devise therapeutic protocols to treat spinal cord injury. These treatments could employ antisense oligonucleotides, antibodies, growth factors, enzymes, pharmaceutical drugs, and gene therapies.
Glial fibrillary acidic protein (GFAP) is the principal intermediate filament protein found in mature astrocytes. Although the exact function of GFAP is poorly understood, it is presumed to stabilize the astrocyte's cytoskeleton and help in maintaining cell shape. Previous studies from our laboratory have shown that when astrocytes were cocultured with primary Schwann cells (pSCs), astrocytes became hypertrophied and fibrous with intensely positive GFAP staining and segregated Schwann cells (SCs) into pockets. In order to understand the functional role of GFAP in this already established astrocyte-SC coculture model, we generated GFAP-negative cell lines from a GFAP-positive astrocytoma cell line and cocultured both the cell lines with pSCs. Our studies demonstrate that the GFAP-positive cell line put out processes toward the SCs, whereas the GFAP-negative cells did not form processes and the majority of the cells remained round. The most significant and interesting finding of this study, however, is the formation of elaborate processes by SCs when grown in coculture with the astrocytoma cells, unlike SCs cultured alone, which showed their typical bipolar spindle-shaped morphology. The extent of processes did not seem to be dependent of GFAP, since SCs cultured with both the cell lines formed similar processes. This coculture model may be useful in elucidating the factor(s) responsible for the formation of processes by SCs and can be further help in our understanding of the mechanism of morphological transformation of SCs.
View details for Web of Science ID A1996VJ82100008
View details for PubMedID 8887943
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.
View details for Web of Science ID A1996UH53300002
View details for PubMedID 8723760
Experimental allergic encephalomyelitis (EAE) is an inflammatory demyelinating disease of the central nervous system (CNS) which has many clinical and pathological features in common with multiple sclerosis (MS). Comparison of the histopathology of EAE and MS reveals a close similarity suggesting that these two diseases share common pathogenetic mechanisms. Immunologic processes are widely accepted to contribute to the initiation and continuation of the diseases and recent studies have indicated that microglia, astrocytes and the infiltrating immune cells have separate roles in the pathogenesis of the MS lesion. The role of cytokines as important regulatory elements in these immune processes has been well established in EAE and the presence of cytokines in cells at the edge of MS lesions has also been observed. However, the role of chemokines in the initial inflammatory process as well as in the unique demyelinating event associated with MS and EAE has only recently been examined. A few studies have detected the transient presence of selected chemokines at the earliest sign of leukocyte infiltration of CNS tissue and have suggested astrocytes as their cellular source. Based on these studies, chemokines have been postulated as a promising target for future therapy of CNS inflammation. This review summarized the events that occur during the inflammatory process in EAE and discusses the roles of cytokine and chemokine expression by the resident and infiltrating cells participating in the process.
View details for Web of Science ID A1996UK62700017
View details for PubMedID 8734446