Several groups have suggested that transplantation of marrow stromal cells (MSCs) promotes functional recovery in animal models of brain trauma. Recent studies indicate that tissue replacement by this method may not be the main source of therapeutic benefit, as transplanted MSCs have only limited ability to replace injured central nervous system (CNS) tissue. To gain insight into the mechanisms responsible for such effects, we systematically investigated the therapeutic potential of MSCs for treatment of brain injury. Using in vitro studies, we detected the synthesis of various growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), and neurotrophin-3 (NT-3). Enzyme-linked immunosorbent assay (ELISA) demonstrated that MSCs cultured in Dulbecco's modified Eagle medium (DMEM) produced substantial amounts of NGF for at least 7 weeks, whereas the levels of BDNF, GDNF and NT-3 remained unchanged. In studies in mice, after intraventricular injection of MSCs, NGF levels were increased significantly in cerebrospinal fluid by ELISA, confirming our cell culture results. Further studies showed that treatment of traumatic brain injury with MSCs could attenuate the loss of cholinergic neuronal immunostaining in the medial septum of mice. These studies demonstrate for the first time that by increasing the brain concentration of NGF, intraventricularly transplanted MSCs might play an important role in the treatment of traumatic brain injury.

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Conventional adenoviral vectors such as E1-deleted first-generation adenovirus (fgAd) elicit striking host immune response, resulting in limited expression of the transgene. A recently described helper-dependent, or gutless, adenoviral vector (hdAd) can promote stable transgene expression in peripheral organs, including the liver. We therefore investigated the safety and durability of hdAd-mediated gene transfer to the central nervous system (CNS) of rats compared with gene delivery by fgAd. Equal amounts of either fgAd or hdAd carrying the beta geo transgene were stereotactically injected into the right hippocampus of adult rats. Transgene expression was assessed by histochemical staining, transgene stability by PCR analysis, and immune infiltration of T lymphocytes and macrophages by immunocytochemical methods. Strong transgene expression from either vector was detected in brain tissue examined on day 6 postinoculation. Thereafter, fgAd-mediated gene expression rapidly decreased, becoming undetectable by day 66, while expression from the hdAd vector persisted throughout the test period. PCR confirmed the presence of hdAd-associated DNA at 66 days postinoculation. The hdAd injection elicited apparently lower numbers of brain-infiltrating macrophages and T cells than did administration of fgAd. These results indicate improved transgene expression and reduced immunogenicity with use of hdAd to deliver genes to the CNS.

We have systematically investigated the therapeutic potenine acetyltransferase (ChAT) activity in cultures following tial of cationic liposome-mediated neurotrophic gene transcalcium-dependent depolarization injury. In in vivo studies, fer for treatment of CNS injury. Following determination of following intraventricular injections of NGF cDNA comoptimal transfection conditions, we examined the effects of plexed with DC-Chol liposomes, ELISA detected nine-to dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) 12-fold increases of NGF in rat CSF. Further studies liposome-mediated NGF cDNA transfection in injured and showed that liposome/NGF cDNA complexes could attenuuninjured primary septo-hippocampal cell cultures and rat ate the loss of cholinergic neuronal immunostaining in the brains. In in vitro studies, we detected an increase of NGF rat septum after traumatic brain injury (TBI). Since deficits mRNA in cultures 1 day after transfection. Subsequent in cholinergic neurotransmission are a major consequence ELISA and PC12 cell biological assays confirmed that culof TBI, our studies demonstrate for the first time that DCtured cells secreted soluble active NGF into the media from Chol liposome-mediated NGF gene transfection may have day 2 after gene transfection. Further experiments showed therapeutic potential for treatment of brain injury. that such NGF gene transfection reduced the loss of chol-