TNF-α production in oligodendrocytes after moderate cervical spinal cord injury

ABSTRACTS / Experimental Neurology 198 (2006) 558 – 597

peripheral organs. Quantitative HS measurements after cell transplant showed significant reduction in the livers of treated mutant mice, mainly in females. In addition, urine bladder distention, usually a major problem in older Naglu mutant mice, was ameliorated only in females. Results suggest that administration of nu-hUCB cells benefits mice modeling Sanfilippo syndrome B; however, sex-based differences in effects need clarification. (Supported by: Lauren’s Hope Foundation; Children’s Medical Research Foundation; Saneron CCEL Therapeutics, Inc. SGD and AEW are consultants and PRS is a co-founder of Saneron CCEL Therapeutics, Inc.). doi:10.1016/j.expneurol.2006.02.086

A novel telomerase-T antigen fusion gene capable of immortalizing human type-1 astrocytes J.F. Sanchez 1, C.J. Schoen 1, L. Shen 2, C.T. Lee 1, J. Chen 1, B.K. Harvey 1, D. Howard 1, Y. Wang 1, J. Shen 2, W.J. Freed 1 1 Cellular Neurobiology Research Branch, NIDA/NIH/DHHS, Baltimore, USA 2 ScienCell Research Laboratories, San Diego, USA Current methodologies for the generation of cell lines involve the use of oncogenic molecules that disrupt cell cycle control through a variety of mechanisms. The immortalization of human cells is specifically complicated by the phenomenon of telomeric shortening, ultimately leading to senescence of after short-term culture. Overexpression of telomerase activity alone in human cells has shown limited promise in compensating for this phenomenon and allowing longer-term propagation. Here we report the generation of a novel immortalizing fusion molecule comprised of a non-transforming, truncated isoform of large T antigen (T155c) and the reverse transcriptase subunit of the human telomerase complex (hTERT). Stable expression of hTERT-T155c fusion protein in primary human type-1 astrocytes derived from 20-week whole brain was able to override cellular senescence, resulting in a 5-day division cycle as demonstrated by cellular proliferation assays. We have passaged a cell population, hA1, which is positive for GFAP and S100b for more than 6 months using nonspecialized medium. In contrast, primary wild-type astrocytes senesce around passage four and fail to retain both glialspecific markers. Immunocytochemical analysis for GABA, 5-HT, ChAT and TH indicates that hA1 cells are capable of neurotransmitter production/uptake. TNFa and IL-1b proinflammatory cytokine sensitivity and subsequent NFkB activation were also found to be intact, as assessed by p65 nuclear translocation. Gene transfer in hA1 cells was efficiently (¨100%) accomplished using AAV2 recombinant viral transduction and although immortal, p53 activity is not compromised in hA1 as indicated by normal p53-dependent reporter activity. Therefore, the telomerase-T155c fusion gene is capable of immortalizing human cells, resulting in cultures with conserved phenotypic properties. Human cell lines produced by this method may therefore prove useful for in

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vitro diagnostic as well as in vivo transplantation studies. (This work was supported by the NIDA(IRP)/NIH/DHHS). doi:10.1016/j.expneurol.2006.02.087

TNF-A production in oligodendrocytes after moderate cervical spinal cord injury S.M. Schaal 1,2, D.D. Pearse 1,2 1 Neuroscience Program, Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, USA 2 Department of Neurological Surgery, Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, USA Tumor necrosis factor alpha (TNF-a), a pro-inflammatory cytokine, is a major cytotoxic molecule present following spinal cord injury (SCI), responsible for initiating/maintaining inflammation and contributing to progressive cell death after trauma. Utilizing an experimental model clinically relevant to the majority of human SCI, this study evaluated the presence of TNF-a after cervical contusion. To induce SCI, Fischer rats were given a moderate cervical contusion using an electromagnetic SCI device (0.95 mm displacement, 20 ms). Temporally, TNF-a mRNA increased within 30 min, peaking at 4 h and returning to normal levels by 24 h based on RT-PCR data. During this time, TNF-a protein increased by 1 h and rose through 6 h as measured by ELISA. Cellular sources of TNF-a rostral to, caudal to, and within the injury epicenter were examined at 1, 4, or 24 h post-injury by immunohistochemistry. Cell-specific markers (NeuN, neurons; CC1, oligodendrocytes) were employed in combination with a TNF-a specific antibody. At all time points evaluated, TNF-a immunoreactivity was present throughout the 1 cm block encompassing the injury epicenter; predominantly in the ventral gray matter and majority of the white matter. However, according to counts performed within each section, the spatial distribution of cellular TNF-a was not significantly different among regions (rostral, epicenter, caudal) within the 1 cm area. Neurons and oligodendrocytes were the principal cellular sources of TNF-a production after moderate cervical SCI. Stereological investigation revealed over 86% of oligodendrocytes stained positive for TNF-a and while neuronal counts were also high, when compared to oligodendrocytes they composed less of the total TNF-apositive cell population. This determination of the cellular source(s) of TNF-a after SCI will enable further elucidation of the mechanisms of its regulation and provide putative therapeutic targets for abating injury-induced cell death. doi:10.1016/j.expneurol.2006.02.088

Transplantation of a ventral mesencephalon-derived cell line, RTC4, reduces ischemic brain damage in rats C.J. Schoen, J.F. Sanchez, B.K. Harvey, O. Dillon-Carter, G.J. Chen, Y. Wang, W.J. Freed, B.J. Hoffer Cellular Neurobiology Research Branch, NIDA/NIH/DHHS, Baltimore, USA