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. © 2005 Wiley‐Liss, Inc.

Gene therapy is considered one of the most promising approaches to developing a novel effective treatment for Pakinson[rsquo]s disease (PD). However, the potential unwanted side effect from viral vector or from the over expression of the transgenes are the major concerns for the future clinical applications. Thus, investigating the optimal vector for gene delivery with a controllable transgene expression mechanism is important. We had reported that helper-dependent adenovirus (hdAd), a novel adenoviral construct with full deletion of viral coding sequences, is an optimal vector to deliver the gene in the lateral ventricle with less immunogenecity and toxicity while offering extended transgene expression (Hum Gene Ther 2001;12). In present study, we injected this vector into the rat striatum to examine the transgene expression profile and the inflammatory response. We also have investigated whether mifepristone-mediated gene switch can be applied in regulating the transgene expression. HdAdv/LacZ vectors were constructed and amplified with the Cre/loxP system as described previously (Mol Ther 2000; 2). Three concentrations of hdAdv/LacZ (1X107, 1X108, and 1X109 particles/ul) were administrated into striatum of rats. After 3days, 7 days, 30days, 60days and 90days post-injection, hdAdv/LacZ expression was quantified by X-gal staining. For the gene switch studies, we have tested the mifepristone-regulated transgene expression in the CNS cell cultures and in animal model study. Different doses of mifepristone were administrated and the transgene expression levels were measured by transgene expression assays. Our results demonstrate that intrastriatal inoculation of hdAdv vector produced a potent transgene expression with minimal inflammatory responses. The hdAdv/lacZ can retrograde transport to the substantia nigra 3-day after intrastriatal injection and last for a long period. The potency of the transgene expression is dose dependent. In in vitro studies, we have found that mifepristone induced a 28-fold higher level of transgene expression in cultures transducted with the gene switch vector, compared with non-mifepristone-treated cultures. The transgene expression levels are mifepristone dose dependent. The results are also confirmed in in vivo study. The preliminary results suggest that mifepristone-induced gene switch system could efficiently induce transgene expression. HdAdv with gene switch system may provide an optimal vector for therapeutic gene delivery for treatment of PD. The next step is to study the therapeutic effects of intrastriatal delivery of hdAdv/GDNF with mifepristone induced gene switch element in PD animal models.

Adult stem cells were once thought to produce only the cell lineages characteristic of the tissues in which they reside. Recent studies suggest that cells derived from one adult tissue can be reprogrammed to change into cellular phenotypes not normally found in that tissue. Bone marrow (BM) derived cells have been demonstrated to differentiate into multiple lineages, including glial cells and neurons, both in vivo and in vitro. This unexpected plasticity of BM cells occurs not only under experimental conditions, but also in humans following BM transplantation. As a result, BM transplantation has emerged as a novel approach to enhance neural regeneration and restore injured brain tissue. Several research teams have reported that transplanted BM cells can differentiate into neural derivatives; indeed, some of these cells were capable of integration into the host brain, where they promoted functional recovery after brain injury. Other researchers conducting similar studies were unable to find any evidence of neural differentiation, concluding that differentiation 'from marrow to brain' is not a common phenomenon. More recently, two papers in Nature also cast doubt on the plasticity of adult stem cells, suggesting that the acquisition of different identities by grafted BM cells may merely reflect their fusion with host cells. Reasons for the wide discrepancies among findings in current BM stem cell research are unclear, making it difficult to understand the mechanisms by which transplanted marrow stem cells provide therapeutic benefit. Here, we summarize recent findings on this subject, and address some of the major controversies that have marked the evolution of adult stem cell research.

Using a cDNA microarray method, we analyzed gene expression profiles in mouse hippocampus after traumatic brain injury (TBI). Of 6,400 randomly selected arrayed genes and expressed sequence tags from a mouse cDNA library, 253 were found to be differentially expressed (106 increased and 147 decreased). Genes involved in cell homeostasis and calcium signaling were primarily up‐regulated while those encoding mitochondrial enzymes, metabolic molecules, and structural proteins were predominantly down‐regulated. Equal numbers of genes related to inflammatory reactions showed increased or decreased expression. Importantly, a large proportion of the dysregulated genes we identified have not been reported as differentially expressed in TBI models. Semiquantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) analyses of representative genes confirmed the validity of the corresponding microarray findings. Thus, our microarray‐based evaluation of gene expression in traumatically injured hippocampus identified both known and novel genes that respond to TBI. Further investigation of these candidate molecules may suggest new ways to attenuate the traumatic effects of brain injury. © 2002 Wiley‐Liss, Inc.

Adenovirus-mediated transfer of the nerve growth factor gene promotes significant recovery of age-related cholinergic neuronal deficits in aged rats, but the effects of such treatment on cognitive dysfunction remain unclear. Herein we report a beneficial effect of first-generation adenovirus-mediated nerve growth factor gene transfer (AdNGF) on the spatial learning and memory of aged rats. The NGF protein was detected by enzyme-linked immunosorbent assay in cerebrospinal fluid as early as 3 days after gene transfer and was expressed for at least 30 days. Escape latency in the Morris water maze hidden-platform test was significantly improved on day 8 postinoculation in memory-impaired rats treated with AdNGF as well as at later testing intervals. Ultimately, the escape latency values for the AdNGF group become indistinguishable from those for aged rats with normal learning capacity. Immunohistochemical analysis of septal cholinergic neurons for choline acetyltransferase (ChAT) showed significant increases in both the number and somal distribution of ChAT-positive cells after inoculation of memory-impaired rats with AdNGF. Improvement in memory performance was positively correlated with increases in both NGF concentration in cerebrospinal fluid (r = 0.73, p = 0.005) and the number of ChAT-staining cells (r = 0.77, p = 0.0022). We conclude that AdNGF can improve cognitive function in memory-impaired aged rats and, with refinements in vector-driven expression of the transgene, may prove suitable for use in humans.

Traumatic brain injury (TBI) causes delayed neuronal deficits that in principle could be prevented by timely intervention with therapeutic genes. However, appropriate vectors for gene transfer to the brain with TBI remain to be developed. First-generation adenoviruses (fgAd) are usually associated with inflammatory and toxic effects when inoculated into brains, despite their high efficiency of gene transfer to these tissues. In this study the authors attempted to determine whether a less immunogenic gene-transfer protocol can be established in the traumatically injured rat brain using helper-dependent adenoviruses (hdAd), a novel adenoviral construct with full deletion of viral coding sequences. Their results show that transgene expression from intrahippocampally inoculated hdAd is maintained for at least 2 months after TBI, in contrast to the much shorter duration of fgAd-mediated gene expression. There was only minimal secretion of proinflammatory IL-1β and TNF-α after inoculation of hdAd. Furthermore, the hdAd-mediated gene expression was associated with less microglial proliferation, astrocytic activation, and macrophage infiltration than observed in fgAd-inoculated brains. There was no additional tissue loss after hdAd inoculation compared with PBS injection. Although both anti-adenoviral and neutralizing antibodies were found in serum after brain inoculation of hdAd, they did not appear to affect transgene expression. The results suggest that hdAd are less immunogenic vectors than conventional adenoviral vectors, and offer improved vehicles for long-term therapeutic transgene transfer to traumatically injured brains.

Hippocampal N‐methyl‐D‐aspartate (NMDA) receptor subunits, by virtue of their involvement in excitotoxic injury as well as memory association, may play an important role in the pathophysiologic mechanisms of traumatic brain injury (TBI). In this study, temporal changes in NMDA receptor subunit (NR1, NR2A, and NR2B) levels in rat hippocampus after TBI were investigated by Western blot and mRNA expression levels by RT‐PCR methods. Sprague‐Dawley rats (250–350 g) were employed, and a controlled cortical impact injury device was used to produce the TBI in rodents. At different postinjury time points (2, 6, 12, 24, and 48 hr), the rat hippocampi were dissected out for protein and RNA preparation. Western blot analysis revealed significant decreases of NR1, NR2A, and NR2B subunit proteins at 6 and 12 hr postinjury in rat hippocampus. Complete recovery of NR1, NR2A, and NR2B subunit protein to the levels of sham controls was observed at 24 hr postinjury. However, RT‐PCR analysis did not show any significant change in the mRNA levels at 2, 6, and 12 hr postinjury in comparison with sham controls, suggesting nontranscriptional change in the levels of these subunits. Thus, TBI can produce transient degradation of NMDA receptor subunits in the hippocampus, which might contribute to temporary memory impairment after injury. © 2002 Wiley‐Liss, Inc.

Transfer of the neurotrophin gene into brain can attenuate age-related deficits such as neuronal atrophy and memory loss, but a suitable vector for this procedure has been lacking. The toxicity and immunogenicity of first-generation adenoviral vectors with E1 deletion (fgAdv) prohibit the application of gene transfer in the majority of central nervous system disorders. Here, we report less toxic and persistent gene expression mediated by helper-dependent adenovirus (hdAdv) in aged rat brain. After intrahippocampal or intraventricular inoculation of the vector, transgene expression was monitored by X-Gal staining and compared with fgAdv-mediated expression. Host inflammatory and immune responses against these vectors were evaluated by immunohistochemical detection of microglia, astrocytes, and infiltrating macrophages, as well as by enzyme-linked immunosorbent assay of cytokines TNF-alpha and IL-1beta. Transgene expression mediated by hdAdv persisted for more than 183 days regardless of inoculation site, as compared with 33 and 66 days for fgAdv-mediated expression after intraventricular and intrahippocampal inoculation, respectively. Inoculation with hdAdv was also associated with reduced numbers of activated microglial cells, astrocytes, and infiltrating macrophages in brain tissue. Secretion of the proinflammatory cytokines TNF-alpha and IL-1beta was minimal after hdAdv but not after fgAdv inoculation. These findings indicate that hdAdv would provide a safe and effective means to transfer therapeutic genes into aged brain.

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

Brain-derived neurotrophic factor (BDNF) has therapeutic potential for treatment of the injured central nervous system. BDNF induces both differentiation and survival of neurons by binding to trkB receptors. This interaction stimulates the intrinsic tyrosine kinase activity of trkB, initiating a signal cascade involving the phosphorylation of intracellular protein on tyrosine, serine, and threonine residues. The purpose of this investigation was to examine the effects of cationic lipid-mediated gene transfection of BDNF on phosphatidylinositol 3 (PI3)-kinase activity in primary septo-hippocampal cell cultures. Thirty-six hours after BDNF gene transfection in the primary CNS cell culture, PI3-kinase activity was significantly increased. The increased PI3-kinase activity was inhibited by wortmannin, a selective and irreversible inhibitor of PI3-kinase. In addition, wortmannin blocked neurofilament increases induced by BDNF gene transfection. This result suggests a possible role of PI3-kinase activation in neuroprotective effects produced by BDNF gene transfection. J. Neurosci. Res. 52:192–200, 1998. © 1998 Wiley-Liss, Inc.

This paper briefly reviews general principles of gene therapy with emphasis on the therapeutic potential of cationic liposome-mediated neurotrophin gene transfer to treat central nervous system (CNS) injury. Current developments in studies of gene therapy for CNS injury are both impressive and promising. Ex vivo gene transfer into the CNS is relatively mature in animal studies following more than a decade of experimental studies. In vivo gene transfer into the CNS has gained more attention recently. Although progress has been made using viral vectors, rapid advances in transfection technologies employing cationic liposomes, together with the relatively low toxicity of these nonviral vector systems, suggest that liposomes may have significant potential for clinical applications. Although many investigators have recognized that gene therapy may be useful for treatment of certain genetic defect diseases or cancer, gene therapy for CNS injury is relatively novel. In contrast to genetic defect disorders, temporary induction of transgenes may have therapeutic applications for CNS injuries such as stroke and trauma. Employing gene transfer techniques to achieve therapeutically useful levels of expression of neurotrophins in the CNS could provide a new strategy for treatment of the traumatically injured CNS.

The blow procedure of a die-forging hammer is analysed theoretically taking account of the elastic deformation of the cushion under the anvil block, and the new formulae for calculating the maximum blow force are deduced from a new model proposed in the paper. The results calculated by the formulae agree well with experimental data. Thus, the formulae and the results provide a basis for designing and using die-forging hammers properly.

WE examined the potential of non-viral vector-mediated gene transfection in the rat spinal cord. Reporter gene (beta-gal) or brain-derived neurotrophic factor (BDNF) cDNA containing a pCMV promoter complexed with DC-Chol liposomes was injected into the intact rat spinal cord gray matter. RT-PCR confirmed the increased expression of BDNF mRNA in the injection areas. X-gal staining demonstrated the localized expression of beta-gal reporter genes. No overt tissue damage caused by DC-Chol liposome/DNA complex injections was detected. These results suggest that cationic liposome-mediated delivery can be a practical method for gene transfer in spinal cord.

Levels of brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) mRNA expression were measured in a rodent model of traumatic brain injury (TBI) following unilateral injury to the cerebral cortex, To obtain reliable data on the co-expression of neurotrophin genes, adjacent coronal sections from the same rat brains were hybridized in situ with BDNF and NT3 cRNA probes, BDNF mRNA increased at 1, 3, and 5 hr after unilateral cortical injury in the cortex ipsilateral to the injury site and bilaterally in the dorsal hippocampus, NT3 mRNA did not change significantly following injury. Our results suggest that TBI produces rapid increases in BDNF mRNA expression in rat brain without changes in NT3 mRNA expression, a finding which differs from studies of ischemia and seizures, It is possible that increased levels of BDNF mRNA rather than NT3 are important components of pathophysiological responses to TBI. (C) 1996 Wiley-Liss, Inc.

We examined the effect of a 6 min depolarization with 60 mM KCl and 1.8, 2.8 or 5.8 mM extracellular CaCl2 on neurofilament proteins of high (NF-H), medium (NF-M) and low (NF-L) molecular weight in primary septohippocampal cultures. One day after depolarization, Western blot analyses revealed losses of all three neurofilament proteins. Increasing the extracellular calcium concentration from 1.8 to 5.8 mM CaCl2 in the presence of 60 mM KCl produced increased losses of all three neurofilament proteins to approximately 80% of control values in the absence of cell death. Calcium-dependent losses of the neurofilament proteins correlated with calcium-dependent increases in calpain 1-mediated breakdown products of alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization and made available to cultures for 24 h, reduced losses of all three neurofilament proteins to approximately 14% of control values. The protective effects of calpain inhibitors 1 and 2 were influenced by different levels of extracellular calcium. Qualitative immunohistochemical evaluations confirmed semiquantitative Western blot data on neurofilament loss and protection by calpain inhibitors 1 and 2. We propose that brief depolarization causes loss of neurofilament proteins, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.