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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Abstract: Adverse effects of cDNA and oligonucleotide delivery methods have not yet been systematically analyzed. We introduce a protocol to monitor toxic effects of two non-viral lipid-based gene delivery protocols using CNS primary tissue. Cell membrane damage was monitored by quantifying cellular uptake of propidium iodide and release of cytosolic lactate dehydrogenase to the culture medium. Using a liposomal transfection reagent, cell membrane damage was already seen 24 hr after transfection. Nestin-positive target cells, which were used as morphological correlate, were severely diminished in some areas of the cultures after liposomal transfection. In contrast, the non-liposomal transfection reagent revealed no signs of toxicity. This approach provides easily accessible information of transfection-associated toxicity and appears suitable for prescreening of transfection reagents.

Lipid-mediated gene transfer (“transfection”) to primary CNS cell cultures is a procedure first introduced by Felgner & Ringold (1989). Since then, different transfection reagents, including liposomes, non-liposomal lipids and activated polyamidoamine dendrimers (Hudde et al. 1999; Mortimer et al. 1999) have been applied to CNS tissue. Liposomal transfection mixtures, including DOSPA/DOPE formulations have been successfully used to transfect various glioma cell lines (Bell et al. 1998), and shown to target cells in CNS explant cultures (Bauer et al. 2001). Furthermore, non-liposomal transfection reagents have served to introduce plasmids coding for therapeutic genes into primary CNS tissue, resulting in improved cell survival and function of the graft within experimental cell replacement therapy (Bauer et al. 2000). Along this line, direct injection of plasmid DNA-liposome complexes into the striato-nigral system has been shown to be sufficient to ameliorate symptoms in parkinsonian rats (Imaoka et al. 1998). More recently, non-viral, liposomal and non-liposomal delivery of oligonucleotides to silence genes by RNA interference (RNAi) have been successfully applied (Davidson & Paulson 2004; Shoji & Nakashima 2004).

Adverse effects of transfection reagents administered systemically are known, e.g. lipid-DNA complexes applied intravenously or by inhalation via the lungs result in inflammatory responses, hence diminishing both transfection yields and transgene expression (Scheule et al. 1997; Tousignant et al. 2000). In contrast, knowledge on toxic effects and tissue damage following local administration of lipid-DNA complexes to various tissues including brain parenchyma is thus far limited.

During the process of optimizing transfection efficiencies we found it necessary to establish an in vitro test system for monitoring both toxic effects and morphological changes after administration of lipid-DNA complexes. This was carried out in organotypic cultures derived from embryonic ventral mesencephalon. Since the cellular environment of organotypic explant cultures closely resembles that found in vivo, exposure of such cultures to transfection mixtures should mimic those conditions found after injection in certain brain areas.

In a comparative approach, we analyzed the cytotoxic effects of a widely used liposomal (lipofectamine) and a non-liposomal (effectene) transfection reagent, suitable to deliver cDNA (Bauer et al. 2000 & 2001) as well as oligonucleotides (Krichevsky & Kosik 2002) to primary tissue/cells. We applied these two conceptually different commercially available transfection reagents on primary mesencephalic organotypic neuronal cultures as our test system. This allowed us to compare reagent-specific toxic effects on primary neuronal target cells, as well as to establish criteria for the prescreening of reagents for local toxicity and tissue damage prior to experimental therapeutic in vivo applications of transfection reagents on mesencephalic tissue.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Culture system. Ventral mesencephalon from embryonic day 15 (E15) Sprague-Dawley rats were isolated and divided into 4 equal-sized tissue blocks (1 mm×1.5 mm×0.5 mm). These were transferred into conical tubes with serum-containing medium consisting of 88.5% RPMI (Gibco), 1.5% glucose (20% solution) and 10% foetal calf serum (Gibco), and then placed in a roller drum within an incubator at 37 ° (Gähwiler 1981). Tissue samples were grown as “free-floating roller tube” (FFRT) cultures as described in detail by Spenger et al. (1994).

Vector and transfection procedure. For transfection experiments, we used a plasmidal vector construct, pCMVeGFP, that codes for enhanced green fluorescence protein and is driven by a cytomegalovirus promoter. Plasmid DNA was purified with the EndoFreeTM Kit (Qiagen) to minimize endotoxine contamination. Optimization of liposome-mediated transfection was conducted by first complexing plasmid DNA (0.1 μg–6 μg) and lipofectamine (Gibco, Karlsruhe, Germany) (0.5 μl–40 μl) in 100 μl Optimem (Gibco), incubating the mixture for 45 min. and then applying it to the cultures for 12 hr (final volume 1 ml). Effectene (Qiagen, Hilden, Germany) was used as a non-liposomal transfection reagent as previously described (Bauer et al. 2000). Briefly, 2 μg DNA were incubated with 16 μl Enhancer (Qiagen) for 5 min., and subsequently complexed with 20 μl effectene in 150 μl Buffer CE (Qiagen) for an additional 10 min. The mixture was then added into the roller tube containing one milliliter medium and incubated for 12 hr. Transfection was done one day after dissection (“day in vitro 1”) (fig. 1).

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Figure 1. Flow diagram. DIV=day in vitro. PI=propidium iodide.

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Indicators of cell membrane damage: Propidium iodide uptake. Propidium iodide (3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide, Sigma) is a stable fluorescent dye, which absorbs blue-green light (493 nm) and emits red fluorescence (630 nm). Being a polar compound, propidium iodide accesses cells via damaged or leaky cell membranes, where upon it interacts with DNA to yield a bright red fluorescence. Propidium iodide is non-toxic to neurones and, due to its already mentioned properties, is a widely used indicator of neuronal membrane integrity and cell damage (Pozzo et al. 1994).

In the present study propidium iodide was used to quantify cell degeneration in ventral mesencephalon tissue blocks after exposure to lipid transfection reagents. We utilized the protocol developed by Kristensen et al. (1999) and Noraberg et al. (1999), whereby 20 μl of 0.1 mM propidium iodide were added to the culture medium to achieve a final concentration of 2 μM. The propidium iodide uptake in the individual cultures was recorded by fluorescence microscopy (Olympus IMT-2, 4X (Splan FL2)), using a standard rhodamine filter and a digital camera (Sensys KAF 1400 G2, Photometrics, Tucson, AZ, USA) at “day in vitro 2, 4 and 6” (fig. 1). The digital photos were analyzed by densitometry using NIH Image 1.62 image analysis program (National Institute of Health, USA) after first outlining the culture area.

Lactate dehydrogenase measurement. Cytosolic lactate dehydrogenase (LDH), released into the culture medium from dead or degenerating cells with permeabilized membranes, was analyzed after “day in vitro 2, 4 and 6” (fig. 1) by spectrophotometry (COBAS MIRA, Roche), according to the method of Vassault (1983). The medium collected for LDH determination was always immediately frozen and stored at −20 ° until analysis. At the start of each session of sample measurements, a standard absorbance curve was determined by first measuring the alterations in absorbance of standard LDH solutions (Boehringer Mannheim). Aliquots of media (20 μl each) were prepared with pyruvate (Sigma) and nicotinamide adenine dinucleotide (NADH; Sigma) in TBS, and the absorbance (340 nm; 37 °) of the reaction mixture was recorded automatically for a period of 120 sec., starting with a lag time of 30 sec. The LDH activity was calculated from the slope of the linear range of the absorbance curve.

Immunohistochemistry. For immunohistochemical investigations, cultures were fixed for 60 min. in 0.1 M phosphate buffer (PB) containing 4% paraformaldehyde. After equilibration in 10% sucrose-PB for 24 hr, cultures were cut in 20 μm slices on a freezing microtome. Visualization of eGFP, nestin, microtubule-associated protein-2, NeuN, tyrosine hydroxylase (TH) and glia fibrillary acidic protein (GFAP) expressing cells was done by immunofluorescence. Sections were washed in 0.1 M phosphate-buffered saline (PBS) for 30 min., incubated in PBS containing 0.3% Triton X-100 and 10% horse serum for 60 min., and then incubated with rabbit anti-GFP antibody (1:800, Invitrogen), mouse anti-nestin (1:1000, Pharmingen), mouse anti-MAP2 (1:2000, Sigma), mouse anti-GFAP (1:800, Chemicon), anti-NeuN or mouse anti-TH (1:500, Chemicon) overnight at 4 °. Sections were washed in PBS and incubated for 2 hr in a biotinylated anti-mouse antibody (1:200, Vector Lab.) and an avidin-biotinylated horseradish peroxidase complex according to the suppliers instructions (Vector Lab.). Finally, immunoreactive cells were visualized by incubation with 0.1% DAB (Pierce) in 0.1 M PB. Sections were dehydrated in ascending alcohol concentrations, cleaned in xylene and cover-slipped. Specificity of immunostaining was determined by omission of primary or secondary antibodies.

Estimation of transfection efficiency. Transfected GFP-positive cells were counted by fluorescence microscopy (Zeiss) after blind coding of the transfection method. In order to be included in the GFP-positive cell count, both dense staining and a clearly defined cell body were required. Estimations of GFP-immunoreactive cells in the tissue blocks were done as previously described (Meyer et al. 1999; Bauer et al. 2000). Cell counts were performed in three, 20 μm thin sections spaced equidistant within 360 μm of the central part of the explants. The total volume of each culture was estimated based on the assumption that the explants were spherically shaped. One individual culture was assumed as one treatment group. Cell counts were corrected according to Abercrombie's formula (Abercrombie 1946). Counts are presented as number of cells per volume.

Statistics. Comparison of two treatment groups (GFP positive cells) was done by means of a t-test for independent means, and comparison of multiple groups (propidium iodide uptake and LDH concentration) was performed by one-way analysis of variance (ANOVA) for independent groups, followed by the Tukey post-hoc test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

Both liposomal (lipofectamine) and non-liposomal (effectene) transfection reagents were used for plasmid transfer (“transfection”) into E15 rat ventral mesencephalon tissue. Transfection protocols were optimized with the aim of maximizing transfection efficiency for each reagent. In this respect, highest transfection rates were obtained when 20 μl lipid solution were complexed with 2 μg plasmid DNA, for both the liposomal and non-liposomal transfection reagents. Transfection yields differed markedly, whereby 3.9 times higher counts of GFP-positive cells (P<0.05) in effectene-transfected (651±199 cells/mm3, n=7) were observed, compared to lipofectamine-transfected ventral mesencephalon tissue (168±94 cells/mm3, n=7) (fig. 2). Given a constant lipid-DNA ratio, doubling DNA amounts (4 μg) in non-liposome-DNA complexes did not alter transfection yields (628±138 cells/mm3, n=7), whereas a considerable decrease in yields was noted for liposome-DNA complexes (33±12 cells/mm3, n=6) (fig. 3). Transfection mixtures with DNA amounts below 1 μg did not result in any detectable transgene expression in both transfection reagents.

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Figure 2. Transfected cells within ventral mesencephalic explant cultures. Ventral mesencephalon tissue derived from E15 rat embryos was transfected with pCMVeGFP at day ion vitro 1. Two μg vector DNA was complexed with 20 μl lipid solution. Figures show transfected cells on the surface of free-floating rooler tube cultures using lipofectamine as a liposomal (A) and effectene as a non-liposomal (B) transfection reagent (scale bar: 300 μm).

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image

Figure 3. DNA concentration and transfection yields. DNA was complexed 1:10 with either effectene (EFF) or lipofectamine (LFA). Ventral mesencephalon cultures were transfected at day in vitro 1 and fixed 5 days post-transfection (day in vitro 5) for evaluation of GFP-positive cells. (#P<0.05 ANOVA, Tukey Test, 1 μg versus 2 μg and 4 μg; +P<0.05 ANOVA, Tukey Test).

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Cellular uptake of propidium iodide was used as an indicator of possible cell membrane damage following exposure to the various transfection mixtures. A significant increase of propidium iodide uptake with exposure time in the culture medium was observed in both transfected and non-transfected control groups (P<0.05) (fig. 4). This time-dependent increase in PI fluorescence is believed to represent primarily non-specific staining of viable cells, an effect that has been also shown in hippocampal slice cultures (Vornov et al. 1998). With liposomal transfection, when cultures were exposed to 2 μg plasmid DNA complexed with 20 μl lipofectamine, propidium iodide uptake was 2.1 times (889±72, n=12 versus 420±17, n=16), 1.9 times (1597±84, n=12 versus 855±74, n=16) and 2.0 times (1772±68, n=12 versus 906±77, n=16) higher than the propidium iodide uptake in non-transfected control groups measured one day, three and five days post transfection, respectively (P<0.05) (fig. 4A).

image

Figure 4. Propidium iodide (PI) uptake in rat ventral mesencephalon tissue after lipid-mediated plasmid transfer. E15 rat ventral mesencephalon tissue was transfected at day in vitro (DIV) 1 with 2 μg plasmid DNA complexed with a liposomal (lipofectamine, LFA) or a non-liposomal (effectene, EFF) transfection reagent, non-transfected cultures served as controls (A). The PI assay was performed one day (DIV 1+1), three days (DIV 1+3) and five days (DIV1+5) post-transfection. Ventral mesencephalon tissue was exposed to Lipofectamine (20 μl), effectene (20 μl) or DNA (2 μg) alone for 12 hr (B). (*P<0.05 ANOVA, Tukey Test, DIV1+1 versus DIV 1+3 and DIV 1+5; #P<0.05 ANOVA, Tukey Test).

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Using non-liposomal transfection, no differences were seen in cultures transfected with effectene-DNA complexes compared to controls (fig. 4A). Doubling the amount of effectene-plasmid DNA did not result in higher propidium iodide uptake, whereas increasing lipofectamine to 40 μl and DNA to 4 μg further increased propidium iodide uptake by up to 42% (data not shown). In separate experiments, ventral mesencephalon explants were also exposed to either liposomes alone, non-liposomal lipids alone or DNA alone, in an attempt to analyze whether the lipids alone or a composite effect of compound mixtures would cause the toxic effects on the target tissue (fig. 4B). Propidium iodide uptake with effectene or plasmid DNA alone (2 μg) developed as with untreated control cultures (compare fig. 4A and B). Effectene alone (20 μl) increased propidium iodide uptake slightly and comparable to the slight increase seen with effectene-DNA mixtures. Lipofectamine alone (20 μl) caused a significant increase in propidium iodide uptake very similar to that seen with lipofectamine-DNA mixtures (P<0.05) (fig. 4B). This strongly suggests that the liposome itself is toxic to the primary cells used here.

Spectophotometric analysis of lactate dehydrogenase (LDH), a marker for cell lysis of culture medium from transfected and non-transfected cultures showed no significant differences between treatment groups and controls (fig. 5).

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Figure 5. LDH measurements in the supernatant of rat ventral mesencephalon tissue. Medium derived from transfected (effectene (EFF)-DNA and lipofectamine (LFA)-DNA) and from non-transfected controls was collected one day in vitro (DIV1+1), three days (DIV1+3) and five days (DIV1+5) post-transfection (no significant differences).

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Immunohistochemical analysis of transfected and non-transfected cultures, for markers of mature neurones (MAP-2, NeuN, TH), glia cells (GFAP) and undifferentiated neuroepithelial precursor cells (nestin), revealed an insolated loss of nestin immunoreactivity in 30% of all sections studied when lipofectamine-DNA had been applied (9 out of 30, fig. 6). No decrease of any cellular marker could be observed in sections taken from effectene-DNA-exposed cultures or control cultures which suggests that this transfection mixture does not impair integrity of the organotypic primary material.

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Figure 6. Nestin-immunoreactive cells in transfected rat ventral mesencephalon tissue. Rat VM tissue was exposed to effectene (EFF)-DNA (A, B) and lipofectamine (LFA)-DNA (C,D) complexes one day post dissection. Ventral mesencephalon tissue was cultured for additional five days prior to immunohistochemical evaluation (left panel low magnification, right panel high magnification; scale bar: 50 μm).

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We studied toxicity of two widely used although chemically different types of transfection reagents in a concentration range applicable for achieving optimal transfection efficiencies in the cell system used (Bauer et al. 2000). The main physico-chemical parameters influencing transfection efficiencies in various lipid-DNA systems are related to the size, stability and charge density of the complexes (Chesnoy & Huang 2000). In addition, there is experimental evidence that the mitotic activity of the target cells, incubation time, as well as the DNA-liposome ratio are crucial factors that affect transfection yields in cell lines and primary tissue (McQuillin et al. 1997; Mortimer et al. 1999). Further, effectene-mediated transfection efficiencies are negatively correlated with increasing lipid to DNA ratios, whereby maximal yields are obtained with 10:1 lipid-DNA ratio and lowest yields with a 50:1 ratio (Bauer et al. 2000). In contrast, liposomal lipofectamine-mediated gene transfer in our cellular system were constant with 2 μg DNA and liposome-DNA ratios in a range of 3:1 to 10:1 (data not shown). This finding may be explained by that transfection efficiencies are more uniform within a broader lipid-DNA ratio range with lipofectamine and other liposomal reagents (McQuillin et al. 1997; Bell et al. 1998).

Toxicity is an often overseen but very critical factor within non-viral gene delivery protocols. In this study toxicity of two transfection methods and their key components were analyzed, using propidium iodide-uptake, release of LDH measurement to the medium and histological evaluation. Propidium iodide is a polar compound, which can only enter cells when cell membranes have lost integrity. The dye has been used as an indicator of neuronal integrity and cell viability (Macklis & Madison 1990). When propidium iodide uptake was quantified and compared, using established standardized protocols in relation to excitotoxic and other toxic damage to brain slice cultures (Noraberg et al. 1999), cell membrane damage above control was detectable by increased propidium iodide uptake as early as 1 day post-transfection after liposome mediated gene transfer, whereas no transfection related changes in propidium iodideuptake were seen in effectene-transfected cultures. For liposomes, a positive correlation of transfection yields and the degree of toxicity in glioma cell lines was observed by Bell et al. (1998) leading to the proposal that some degree of cell membrane damage is required for optimal transfection, bearing a fine margin between optimized transfection and cytotoxicity. However, effectene-DNA complexes successfully transfect cells to a higher degree without causing measurable effects on the cell membrane what might be due to a different biophysical mechanism regarding the entry of the complexes into the cytosol. Interestingly, our data show that the liposomal lipid compound alone is sufficient to cause cell membrane damage, pointing to a general adverse effect of this reagent rather than specific toxicity due to uptake or processing of lipid-DNA complexes with a specific structure, size or charge.

Immunohistochemical investigations revealed areas with a selective loss of nestin immunoreactive cells, considered as the main targets of lipid mediated gene transfer (Bauer et al. 2000 & 2001), in lipofectamine-transfected cultures. Immunohistochemical analysis at 3 days post-transfection did show some fading of nestin immunoreactivity in some cells but no total loss as observed 5 days post-transfection was seen.

There was a tendency of LDH levels to increase in lipofectamine-transfected cultures, indicating delayed substantial damage of the target cells. Nevertheless, nestin-positive cells represent only a small subset of cells within the organotypic ventral mesencephalon culture and even substantial loss of nestin positive cells will be accompanied with only moderate increase of LDH in the supernatant. Thus, changes in propidium iodide uptake is the earliest and most sensitive marker to assay transfection-mediated impairment of cellular integrity in our test system.

Conclusion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We have introduced the use of foetal brain explants in combination with markers of cell death for testing the toxicity of liposomal and non-liposomal transfection reagents. Using standardized protocols to quantify the cellular uptake of propidium iodide and LDH release in the culture medium impairment of cell membrane integrity was the first detectable sign of transfection-associated toxicity, followed by morphological/immunohistochemical changes, which were indicators for more extensive and later damage in this system. Lactate dehydrogenase measurements seem to be a less sensitive marker for cell-membrane damage in this system, although including it might be useful to quantify more extensive membrane-and cell-damage. We anticipate that this test system can be more generally applied to get a better understanding of transfection-associated toxicity of different reagents and to prescreen transfection reagents prior to in vivo applications into the striato-nigral system in the brain.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References

We thank D. Lyholmer for technical assistance and Dr. U. Olazabal for preparing the manuscript. Professor G.W. Bornkamm and Professor T. Meitinger for continuously supporting the project. This research was supported by DFG (Nr. UE59/21) and the Danish MRC.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Conclusion
  7. Acknowledgements
  8. References
  • Abercrombie, M.: Estimation of nuclear population from microtome sections. Anat. Rec. 1946, 94, 239247.
  • Bauer, M., M. Meyer, L. Grimm, T. Meitinger, J. Zimmer, T. Gasser, M. Ueffing & H. R. Widmer: Non-viral GDNF gene transfer enhances survival of cultured dopaminergic neurons and improves their function after transplantation in a rat model of Parkinson's disease. Hum. Gene Therapy 2000, 11, 15291541.
  • Bauer, M., M. Meyer, J. Sautter, T. Gasser, M. Ueffing & H. R. Widmer: Liposome-mediated gene transfer to fetal human ventral mesencephalic explant cultures. Neurosci. Lett. 2001, 308, 169172.
  • Bell, H., W. L. Kimber, M. Li & I. R. Whittle: Liposomal transfection efficiency and toxicity on glioma cell lines: in vitro and in vivo studies. NeuroReport 1998, 9, 793798.
  • Chesnoy, S. & L. Huang: Structure and function of lipid-DNA complexes for gene delivery. Annu. Rev. Biophys. Biomol. Struct. 2000, 29, 2747.
  • Davidson, B. L. & H. L. Paulson: Molecular medicine for the brain: silencing of disease genes with RNA interference. Lancet Neurology 2004, 3, 145149.
  • Felgner, P. L. & G. M. Ringold: Cationic liposome-mediated transfection. Nature 1989, 337, 387388.
  • Gähwiler, B. H.: Organotypic monolayer cultures of nervous tissue. J. Neurosci. Meth. 1981, 4, 329342.
  • Hudde, T., S. A. Rayner, R. M. Comer, M. Weber, J. D. Isaacs, H. Waldmann, D. F. Larkin & A. J. George: Activated polyamidoamine dendrimers, a non-viral vector for gene transfer to the corneal endothelium. Gene Ther. 1999, 6, 939943.
  • Imaoka, T., I. Date, T. Ohmoto & T. Nagatsu: Significant behavioral recovery in Parkinson's disease model by direct intracerebral gene tranfer using continuous injection of a plasmid DNA-liposome complex. Hum. Gene Therapy 1998, 9, 10931102.
  • Krichevsky, A. M. & K. S. Kosik: RNAi functions in cultured mammalian neurons. Proc. Nat. Acad. Sci. USA 2002, 99, 1192611929.
  • Kristensen, B. W., J. Noraberg, B. Jakobsen, J. B. Gramsbergen, B. Ebert & J. Zimmer: Excitotoxic effects of non-NMDA receptor agonists in organotypic corticostriatal slice cultures. Brain Res. 1999, 841, 143159.
  • Macklis, J. D. & R. D. Madison: Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. J. Neurosci. Meth. 1990, 31, 4346.
  • McQuillin, A., K. D. Murray, C. J. Etheridge, L. Stewart, R. G. Cooper, P. M. Brett, A. D. Miller & H. M. Gurling: Optimization of liposome mediated transfection of a neuronal cell line. NeuroReport 1997, 8, 14811484.
  • Meyer, M., J. Zimmer, R. W. Seiler & H. R. Widmer: GDNF increases the density of cells containing calbindin but not of cells containing calretinin in cultured rat and human fetal nigral tissue. Cell Transplant. 1999, 1, 12.
  • Mortimer, I., P. Tam, I. MacLachlan, R. W. Graham, E. G. Saravolac & P. B. Joshi: Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Therapy 1999, 6, 403411.
  • Noraberg, J., B. W. Kristensen & J. Zimmer: Markers for neuronal degeneration in organotypic slice cultures. Brain Res. Protocols 1999, 3, 278290.
  • Pozzo, N., L. D. Miller, N. K. Mahanty, J. A. Connor & D. M. Landis: Spontaneous pyramidal cell death in organotypic slice cultures from rat hippocampus is prevented by glutamate receptor antagonists. Neuroscience 1994, 63, 471487.
  • Scheule, R. K., J. A. St. George, R. G. Bagley, J. Marshall, J. M. Kaplan, G. Y. Akita, K. X. Wang, E. R. Lee, D. J. Harris, C. Jiang, N. S. Yew, A. E. Smith & S. H. Cheng: Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung. Hum. Gene Therapy 1997, 8, 689707.
  • Shoji, Y. & H. Nakashima: Current status of delivery systems to improve efficacy of oligonucleotides. Curr. Pharm. Des. 2004, 10, 785796.
  • Spenger, C., L. Studer, L. Evtouchenko, M. Egli, J. M. Burgunder, R. Markwalder & R. W. Seiler: Long-term survival of dopaminergic neurones in free-floating roller tube cultures of human fetal ventral mesencephalon. J. Neurosci. Meth. 1994, 54, 6373.
  • Tousignant, J. D., A. L. Gates, L. A. Ingram, C. L. Johnson, J. B. Nietupski, S. H. Cheng, S. J. Eastman & R. K. Scheule: Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice. Hum. Gene Therapy 2000, 11, 24932513.
  • Vassault, A.: Lactate dehydrogenase, UV-method with pyruvate and NADH. In: Methods of enzymatic analysis. Eds.: H. U.Bergmeyer, J.Bergmeyer & M.Grossl. , 1993, pp. 118126.
  • Vornov, J. J., J. Park & A. G. Thomas: Regional vulnerability to endogenous and exogenous oxidative stress in organotypic hippocampal culture. Exp. Neurol. 1998, 149, 109122.