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Background: Diabetes increases the risk of certain types of cancer. However, the literature regarding the incidence of diabetes after cancer diagnosis is inconsistent. We aimed to assess whether there was a higher incidence of diabetes among cancer patients by performing a systematic review and meta-analysis of results from cohort studies. Methods: A systematic electronic literature search was carried out from cohort studies regarding the incidence of diabetes in cancer patients, using the databases PubMed (MEDLINE), Embase, Web of Science, and the Cochrane Library. Random-effects meta-analyses were conducted to pool the estimates. Results: A total of 34 articles involving 360,971 cancer patients and 1,819,451 cancer-free controls were included in the meta-analysis. An increased pooled relative risk (RR) of 1.42 (95% confidence interval (CI): 1.30–1.54, I2 = 95, τ2 = 0.0551, p < 0.01) for diabetes in cancer patients was found compared with the cancer-free population. The highest relative risk was observed in the first year after cancer diagnosis (RR = 2.06; 95% CI 1.63–2.60). Conclusions: New-onset diabetes is positively associated with cancer, but this association varies according to cancer type. More prospective studies with large sample sizes and longer follow-up times are advocated to further examine the association and the underlying mechanisms.

Purpose Artificial intelligence (AI) plays a substantial role in many domains, including medical fields. However, we still lack evidence to support whether or not cancer patients will accept the clinical use of AI. This research aims to assess the attitudes of Chinese cancer patients toward the clinical use of artificial intelligence in medicine (AIM), and to analyze the possible influencing factors. Patients and methods A questionnaire was delivered to 527 participants. Targeted people were Chinese cancer patients who were informed of their cancer diagnosis. Results The effective response rate was 76.3% (402/527). Most cancer patients trusted AIMs in both stages of diagnosis and treatment, and participants who had heard of AIMs were more likely to trust them in the diagnosis phase. When an AIM's diagnosis diverged from a human doctor' s, ethnic minorities, and those who had received traditional Chinese medicine (TCM), had never received chemotherapy, were more likely to choose "AIM", and when an AIM's therapeutic advice diverged from a human doctor's, male participants, and those who had received TCM or surgery, were more likely to choose "AIM". Conclusion Most Chinese cancer patients believed in the AIM to some extent. Nevertheless, most still thought that oncology physicians were more trustworthy when their opinions diverged. Participants' gender, race, treatment received, and AIM related knowledge might influence their attitudes toward the AIM. Most participants thought AIM would assist oncology physicians in the future, while little really believed that oncology physicians would completely be replaced.

Population spatial data can more truly express the actual distribution characteristics of the population, and provide data support for the regional environment and population development. Use Shandong Province as the research area, township-level census data, revised DMSP/OLS night-time data, and Globaland30 land cover data as data sources, partitions based on population agglomeration, and uses a stepwise regression method to build a population data spatial model. Use the model to simulate population density with a resolution of 100m. The experimental results show: Stepwise regression model good precision, the average relative error was 23.56%, and Root Mean Square Error, Mean Absolute Error are better than the other two public datasets. The simulation results are better than the two public datasets.

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.

Therapy for Parkinson's disease (PD), a common neurological disorder characterized by pathological degeneration of the nigrostriatal dopaminergic system, remains unsatisfactory. Gene therapy is considered one of the most promising approaches to developing a novel effective treatment for PD. Among the numerous candidate genes that have been tested as therapeutic agents, those encoding tyrosine hydroxylase, guanosine triphosphate cyclohydrolase I and aromatic L-amino acid decarboxylase all boost dopamine production, while glial cell line-derived neurotrophic factor promotes the survival of dopaminergic neurons and is generally believed to possess the greatest potential for successful restoration of the dopaminergic system. The genes encoding vesicular monoamine transporter-2 and glutamic acid decarboxylase have also produced therapeutic effects in animal models of PD. Both viral and non-viral vectors, each with its particular advantages and disadvantages, have been used to deliver these genes into the brain. Whether or not regulatable expression systems are essential to successful gene therapy for PD remains a critical issue in the clinical application of this emerging treatment. Here we review the current status of gene therapy for PD, including the application of control systems for transgene expression in the brain.

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.

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.

Telomerization, the process of immortalization of normal cells by expression of telomerase reverse transcriptase (TERT), could be of great use in biomedicine if the process allows cells to retain their normal properties and does not promote neoplastic transformation. In this article, we review the data on the potential uses of telomerized cells in ex vivo gene therapy, and discuss the issue of the potential risks of the use of this technology. We present preliminary data on the transplantation of telomerized bovine adrenocortical cells in the rat brain. Like other cell types, adrenocortical cells may be engineered to secrete desirable gene products. Currently, problems of immune rejection limit the usefulness of this potential therapy. We discuss future improvements in this cell transplantation system that could address these questions. Telomerization, by removing the senescence barrier to unlimited cell proliferation, will greatly aid the genetic modification of cells in order to solve the issue of immun...

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 PI3kinase activation in neuroprotective effects produced by BDNF gene transfection. J. Neurosci. Res. 52:192‐ 200, 1998. r 1998 Wiley-Liss, Inc.

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

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.

We examined the effect of cationic lipid-mediated gene transfection of nerve growth factor (NGF) in primary septo-hippocampal cell cultures. Rat NGF cDNA was subcloned into a pUC19-based plasmid containing a CMV promoter. Two days after NGF gene transfection in primary cell cultures, ELISA confirmed increases in NGF protein secretion from transfected cells. To study the biological effect of cationic lipid-mediated NGF gene transfection, we analyzed the amount of neurofilament protein from NGF-transfected cell cultures. Western blot and immunohistochemical analyses detected significant increases in the phosphorylated form of neurofilament proteins in the cultures after cationic lipid-mediated NGF cDNA transfection. Cationic lipid-mediated NGF cDNA transfection did rot cause significant changes in the total amount of neurofilament protein. Our studies suggest that cationic lipid-mediated NGF gene transfection can increase neurofilament phosphorylation but not total neurofilament protein.

This paper reviews important new evidence indicating that traumatic brain injury can produce more widespread derangements to the neuronal cytoskeleton than previously recognized. Although cytoskeletal derangements in axons have long been documented, recent data suggest that traumatic brain injury can produce structural derangements to dendrites and cell bodies as well. Many of these investigations have employed in vivo models to provide important insights into mechanisms possibly mediating the acute loss of cytoskeletal proteins, including disturbances in calcium homeostasis and activation of calcium-dependent proteolytic enzymes. However, we have little understanding of processes mediating the recovery of cytoskeletal proteins following injury. This paper provides recent evidence from in vitro models of central nervous system injury that neurotrophic proteins can enhance the recovery of the neuronal cytoskeleton. Neurotrophin-based therapy could employ either administration of exogenous neurotrophic proteins and/or transfection of cDNA for appropriate neurotrophins.

We have previously shown that spatial memory changes following experimental traumatic brain injury (TBI) include long-term changes that are (1) ‘overt’: detected by routine behavioral assessments, or (2) ‘covert’: undetected in the absence of a secondary pharmacological challenge, such as by the cholinergic antagonist, scopolamine. Our objective in this study was to extend this finding by characterizing the time course of recovery of overt and covert spatial memory performance following two magnitudes of experimental TBI. The Morris water maze was used to assess cognitive performance. Rats received either moderate magnitude (6 m/s, 1.7 mm deformation) or low magnitude (6 m/s, 1 mm deformation) impacts through a lateral craniectomy under isoflurane anesthesia. Sham rats underwent identical surgical procedures but were not injured. To avoid motor deficits, water maze testing started two weeks post-injury. Rats were given four trials per day for seven consecutive days. For each trial, latency to find a hidden platform was timed. On the sixth, rats were injected (i.p.) with scopolamine (1 mg/kg) 15 min prior to maze testing. The next day, rats were retested. This testing regimen was repeated, beginning 4, 6, and 10 weeks post-TBI. Results showed that, while the low-magnitude injury produced no overt spatial memory deficits, the moderate-magnitude group exhibited overt deficits during the first test regimen. Also, while both injury magnitudes produced an enhanced sensitivity to spatial memory impairment by scopolamine at two weeks post-TBI, this covert deficit persisted only in the severe group at 4, 6, and 10 weeks post-TBI. Qualitative light microscopy showed that both injury groups had graded cortical necrosis. However, underlying subcortical structures such as the hippocampus appeared intact, with no overt cellular or parenchymal damage to the neuropil. These data suggest three distinct stages of functional recovery: (1) the initial period when overt deficits are present, (2) a period following recovery from overt deficits within which covert deficits can be reinstated by a pharmacological challenge, and (3) a period following recovery from both overt and covert deficits. Covert deficits can persist long after the recovery of overt deficits and, like other neurological deficits, the rate of recovery is dependent on the magnitude of TBI. Finally, spatial memory deficits can occur in the absence of light microscopic evidence of cell death in the hippocampus.

This study employed in vivo microdialysis in awake, freely-moving Sprague-Dawley rats to examine acetylcholine (ACh) release in the dorsal hippocampus at 14 days following lateral controlled cortical impact. Extracellular levels of ACh were measured prior to and after an intraperitoneal administration of scopolamine (1 mg/kg), which evokes ACh release by blocking autoreceptors. At 14 days post injury there were no differences in basal ACh levels. However, injury produced a significant reduction in scopolamine-evoked ACh release. The data suggest that cholinergic neurotransmission remains chronically compromised, and thus may contribute to previously documented post traumatic spatial memory deficits.

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabelling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated)in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Acute biochemical consequences of moderate traumatic brain injury (TBI) include activation of kinases, including protein kinase C (PKC). To determine the possible consequences of PKC activation at the substrate level, we have examined protein phosphorylation patterns 1 h following injury. Although the phosphorylation of most proteins remained unchanged following injury, we observed a significant increase in the phosphorylation of a 61,000 dalton protein (TBI61) in injured rat hippocampus (121% higher than sham control) in vitro. TBI61 phosphorylation could be enhanced by phosphatidyl serine and diacylglycerol or by addition of exogenous PKC. In addition, TBI61 phosphorylation was inhibited by the PKC inhibitor, staurosporine, suggesting further that this protein may be a PKC substrate. These data suggest that TBI increases the phosphorylation of a 61 kD hippocampal protein in vitro. Increases in the protein level and activity of PKC could contribute to this increased phosphorylation.

Calcium-dependent excitotoxic processes contribute significantly to pathologic responses to traumatic brain injury (TBI). TBI causes neuronal depolarization and excessive excitatory neurotransmitter release, which may lead to increases in intracellular calcium levels. However, responses of calcium-dependent enzymes such as protein kinase C (PKC) following TBI are poorly understood. Since PKC plays an important role in signal transduction and maintenance of normal neuronal function, we investigated changes in PKC activity and protein levels following fluid percussion brain injury in rats. We observed a 23.1% increase in PKC activity 1 h postinjury and 80.7% increase in PKC activity 3 h postinjury. There was no statistically significant change in PKC activity 5 min and 24 h after injury. PKC immunolabelling studies detected a significant increase in PKC levels in membrane fractions 3 h but not 1 h after injury. Thus PKC activation is transiently increased following TBI and may play an important role in pathophysiologic responses to TBI.

We examined microtubule-associated protein 2 (MAP2) levels in hippocampal and cortical tissue 3 h following moderate traumatic brain injury (TBI) in the rat. MAP2 levels were assayed by quantitative immunoreactivity in tissue fractions obtained from naive, sham-injured, or fluid percussion-injured animals. Tissues were homogenized in the presence of protease inhibitors (0.3 mM phenylmethylsulfonyl fluoride, PMSF), a specific calpain inhibitors (0.1 mM leupeptin), and chelators (2 mM ethylene glycol-bis-tetraacetic acid, EGTA; 1 mM ethylenedinitrilo-tetraacetic acid, EDTA) to eliminate in vitro MAP2 proteolysis during tissue processing. Compared to naive rats, sham injury had no effect on soluble MAP2 levels in either cortex (105.0 +/- 4.4% of naive value) or hippocampus (106.6 +/- 5.2% of naive value). However, TBI caused a significant (p0.005) decrease in hippocampal MAP2 levels (55.7 +/- 5.9% of sham-injured controls). The effect appeared to be regionally selective, since the MAP2 decrease did not occur in cortex (89.1 +/- 1.4%). The degree of MAP2 decrease in hippocampus was similar in both membrane (57.8%) and cytosolic (55.7%) fractions, ruling out the possibility of partitioning artifacts. The data suggest that sublethal alterations of neuronal structure and function caused by MAP2 degradation may play an important role in the development of TBI-induced functional deficits. Since MAP2 is exclusively associated with the cytoskeleton in somal and dendritic compartments of neurons, the pathophysiology of sublethal magnitudes of TBI may also involve dendritic and somal dysfunction.

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.

Top of pageAbstract 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-1 and TNF- 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. Keywords: Adenovirus, Helper-dependent, Transgene, Immune response, Brain, Trauma

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.

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 βgeo 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.

Exogenous insulin-like growth factor-I (IGF-I) is known to improve the pathophysiology of a thermal injury, however, deleterious side-effects have limited its utility. Cholesterol-containing cationic liposomes that encapsulate complementary DNA (cDNA) are nonviral carriers used for in vivo gene transfection. We propose that liposome IGF-I gene transfer will accelerate wound healing in burned rats and attenuate deleterious side-effects associated with high levels of IGF-I. To test this hypothesis IGF-I gene constructs, encapsulated in liposomes, were studied for their efficacy in modulating the thermal injury response. Thirty adult male Sprague-Dawley rats were given a 60% TBSA scald burn and randomly divided into three groups to receive weekly subcutaneous injections of liposomes plus the lacZ gene coding for beta-galactosidase, liposomes plus cDNA for IGF-I and beta-galactosidase or liposomes plus the rhIGF-I protein. Body weights and wound healing were measured. Muscle and liver dry/wet weights and IGF-I concentrations in serum, skin and liver were measured by radioimmunoassay. Transfection was confirmed by histochemical staining for beta-galactosidase. Rats receiving the IGF-I cDNA constructs exhibited the most rapid wound re-epithelialization and greatest increase in body weight and gastrocnemius muscle protein content (P < 0.05). Local IGF-I protein concentrations in the skin were higher when compared to liposomes containing only the lacZ gene (P < 0.05) Transfection was apparent in the cytoplasm of myofibroblasts, endothelial cells and macrophages of the granulation tissue. Liposomes containing the IGF-I gene constructs proved effective in preventing muscle protein wasting and preserving total body weight after a severe thermal injury.

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.

Liposome-mediated NGF transfection has been shown to increase the expression and secretion of NGF in primary rat septo-hippocampal cell cultures. Here we report that along with increased NGF expression, the activity of choline acetyltransferase, the synthetic enzyme for acetylcholine, is increased by 18% within 2 days, by 41% within 4 days and by 32% within 8 days after NGF gene transfection in septo-hippocampal cell cultures. This result further confirms that biologically active NGF is functionally expressed in septo-hippocampal cells when transfected with cDNA for NGF completed with liposomes.

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.

WE examined the temporal profile of the expression of brain-derived neurotrophic factor (BDNF) cDNA containing a viral promoter following the injection of liposome cDNA complexes into the intact and traumatically injured rat brain. In situ hybridization and PCR confirmed the presence of injected BDNF cDNA for at least 6 days after injection. A similar profile of BDNF cDNA was observed when it was injected following cortical impact injury. mRNA was also localized around the injection areas. These results suggest that liposome-mediated delivery of neurotrophin cDNA may be a practical gene transfer method for treating traumatic brain injury.

Nuclear factor-kappa B (NF-kappa B) DNA binding factor is an inducible transcription factor that responds to various cellular signals. Levels of cortical NF-kappa B DNA binding activity were measured in a controlled lateral cortical impact model of traumatic brain injury (TBI) in rats. Using electrophoretic mobility shift assays (EMSAs), we found that NF-kappa B DNA binding activity in cerebral cortex ipsilateral to the injury site increased at 1, 3, 5 and 7 days after injury. Binding activity peaked at 3 days after injury and subsided by 10 days after injury. These data indicate that TBI produces transient increases in NF-kappa B DNA binding activity. Further insights into the role of NF-kappa B in TBI may provide new therapeutic opportunities for head trauma.

Although liposomes have been widely employed to transfect DNA into a variety of cell types, no previous studies have systematically examined conditions producing optimal liposomal-mediated transfection of DNA into central nervous system (CNS) cells. Thus, we used the beta-galactosidase (beta-gal) reporter gene to examine factors influencing the efficiency of liposome-mediated gene transfection in CNS cell cultures. Our results indicate that without increasing the amounts of DNA, increased liposome concentrations within certain limits enhanced transfection efficiency. However, higher liposome levels could produce cell lysis. Without increasing liposome concentrations, increased amounts of DNA did not improve transfection efficiency. Employing the optimal concentration (1 microgram DNA/3 microliters liposomes/well), beta-gal gene expression was sustained for at least two weeks after transfection in primary septo-hippocampal cultures.

We examined liposome-mediated gene transfection of nerve growth factor (NGF) in primary central nervous system cultures. RT-PCR analyses detected increased expression of NGF mRNA one day after liposome-mediated NGF gene transfection. ELISA studies detected large increases in NGF protein in cells and in culture medium after NGF gene transfection. Cells continued to secrete NGF into the medium for at least 2 weeks. NGF bioassays confirmed that the NGF secreted after gene transfection was biologically active.

Traumatic brain injury (TBI) produces a tissue-specific decrease in protein levels of microtubule-associated protein 2 (MAP2), an important cross-linking component of the neuronal cytoskeleton. Because moderate brain hypothermia (30°C) reduces certain neurobehavioral deficits produced by TBI, we examined the efficacy of moderate hypothermia (30°C) in reversing the TBI-induced loss of MAP2 protein. Naive, sham-injured, and moderate (2.1 atm) fluid percussion-injured rats were assessed for MAP2 protein content 3 h post injury using quantitative immunoreactivity measurements. Parallel groups of sham-injured and fluid percussion-injured animals were maintained in moderate hypothermia (30°C), as measured by temporalis muscle temperature, for MAP2 quantitation 3 h post injury. No difference in MAP2 levels was observed between naive and sham-injured normothermic animals. Hypothermia alone had no effect on soluble MAP2 levels in sham-injured animals compared with normothermic sham-injured controls (88.0 ± 7.3%; p > 0.10). Fluid percussion injury dramatically reduced MAP2 levels in he normothermic group (44.3 ± 5.9%; p < 0.0005) compared with normothermic sham-injured controls. No significant reduction of MAP2 was seen in the hypothermic injured group (95.2 ± 4.6%; compared with hypothermic sham-injured controls, p > 0.20). Although it is premature to infer any causal link, the data suggest that the attenuation of injury-induced MAP2 loss by hypothermia may contribute to its overall neuroprotective action.