(Hypertension. 1999;34:756-761.)
© 1999 American Heart Association, Inc.
Scientific Contributions |
Adenovirus-Mediated Gene Delivery to Hypothalamic Magnocellular Neurons in Mice
From the Departments of Psychology, Pharmacology, and Medicine, and the Cardiovascular Center (T.G.B., A.K.J.), University of Iowa, Iowa City; and the Department of Physiological Sciences (E.C.V., S.S.M.), Biomedical Center, UFES, Vitoria, ES, Brazil.
Correspondence to Alan Kim Johnson, PhD, Department of Psychology, University of Iowa, 11 Seashore Hall E, Iowa City, IA 52242-1407. E-mail alan-johnson{at}uiowa.edu
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Abstract |
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Abstract—Vasopressin is synthesized by magnocellular neurons in supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei and released by their axon terminals in the neurohypophysis (NH). With its actions as an antidiuretic hormone and vasoactive agent, vasopressin plays a pivotal role in the control of body fluids and cardiovascular homeostasis. Because of its well-defined neurobiology and functional importance, the SON/PVN-NH system is ideal to establish methods for gene transfer of genetic material into specific pathways in the mouse central nervous system. In these studies, we compared the efficiency of transferring the gene lacZ, encoding for ß-galactosidase (ß-gal), versus a gene encoding for green fluorescent protein by using replication-deficient adenovirus (Ad) vectors in adult mice. Transfection with viral concentrations up to 2x107 plaque-forming units per coverslip of NH, PVN, and SON in dissociated, cultured cells caused efficient transfection without cytotoxicity. However, over an extended period of time, higher levels (50% to 75% of the cells) of ß-gal expression were detected in comparison with green fluorescent protein (5% to 50% of the cells). With the use of a stereotaxic approach, the pituitary glands of mice were injected with Ad (4x106 plaque-forming units). In material from these animals, we were able to visualize the expression of the ß-gal gene in the NH and in magnocellular neurons of both the PVN and SON. The results of these experiments indicate that Ad–Rous sarcoma virus promoter–ß-gal is taken up by nerve terminals at the injection site (NH) and retrogradely transported to the soma of the neurons projecting to the NH. We conclude that the application of these experimental approaches will provide powerful tools for physiological studies and potential approaches to deliver therapeutic genes to treat diseases.
Key Words: mice • vasopressin • hypothalamus • fluorescence • gene transfer
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Introduction |
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Nonreplicating, recombinant adenovirus (Ad) vectors have been widely used to deliver foreign genes to cells because these vectors are adept at entering cells, transferring their genetic material, and taking over the cellular machinery of the host to synthesize viral proteins.1 2 3 The importance of Ad-mediated gene transfer to the nervous system, where gene expression can modulate or restore the function of target cells, has been recognized.2 4
The contribution of magnocellular hypothalamic neurons located in the supraoptic (SON) and paraventricular (PVN) nuclei in the control of body fluid and cardiovascular homeostasis has been described.5 6 7 8 9 10 The soma of magnocellular neurons in hypothalamic SON and PVN synthesizes the neurohormone vasopressin. Axons from these cells project to the neurohypophysis (NH), where this peptide is stored in nerve terminals for release into the peripheral circulation.5 8
In the present series of studies, we investigated the efficacy of in vitro Ad transfections of mouse SON, PVN, and NH to induce expression of the gene products of lacZ (ß-galactosidase, or ß-gal) and of green fluorescent protein (GFP). The ultimate goal of this study was to determine whether directly transfecting the NH of adult mice with Ad would allow targeted gene delivery to the magnocellular neurons in the PVN and SON via retrograde transport.
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Methods |
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The construction of replication-deficient, serotype 5–derived human Ad vector expressing GFP or the nuclear-targeted Escherichia coli ß-gal by the Gene Transfer Vector Core Laboratory at the University of Iowa, Iowa City, has been described.11 12 In brief, vectors deleted of sequences in the E1A and E1B region, which impair the ability of this virus to replicate and transform nonpermissive cells, were cotransfected into human embryonic kidney (HEK293) cells that complement E1 early viral promoters. A Rous sarcoma virus (RSV) promoter was used to drive transcription of lacZ or gfp with a simian virus 40 polyadenylation sequence. Purified virus (AdRSVß-gal or AdRSVgfp) was prepared by CsCl gradient centrifugation followed by overnight dialysis against 3% sucrose phosphate-buffered saline (PBS). Viral vectors [2x1010 plaque-forming units (pfu)/mL] were stored at -80°C until use.
Experiments were conducted with male and female adult C57BL/6J mice (The Jackson Laboratory, Bar Harbor, Me) weighing 15 to 20 g. The animals were caged individually and had free access to food and water. Room temperature was maintained at 23±0.2°C, and a 12:12-hour light/dark cycle was in effect. All experimental protocols conformed to the National Institutes of Health guidelines for animal research and were approved by the Animal Care and Use Committee at the University of Iowa.
Gene Transfer
For in vitro gene transfer, tissue dissociation and cell culture
were performed using procedures previously reported for studies in rat
pups.13 For each set of cultures, 6 mice were
anesthetized with methoxyflurane (Metofane, Schering-Plough
Animal Health) and decapitated, and the PVN, SON, and NH were
selectively removed. Tissue was digested by dispase I (1.5 U/mL;
Boehringer Mannheim) followed by mechanical trituration to
ensure complete dissociation. The cells were plated onto 18-mm glass
coverslips, and the cultures were maintained in an incubator (Heraeus
Instruments) with a humidified atmosphere of 5%
CO2 and 95% air at 37°C for 3 hours. The cells
were then either inoculated with AdRSVß-gal or
AdRSVgfp or treated with saline–3% sucrose vehicle for 30
minutes, and the medium was replaced. Two thirds of the medium was
replaced every 2 days.
For in vivo gene transfer, mice were anesthetized with pentobarbital (35 mg/kg IP; Abbott Laboratories), and the heads were secured in a stereotaxic instrument (David Kopf Instruments). With the use of sterile surgical techniques, the skull was leveled between the bregma and lambda after a longitudinal incision of the scalp and cleansing of the exposed dorsal cranium. The stereotaxic coordinates were 2.3 mm caudal to the zero coordinate (the intersection between the anterior coronal and sagittal sutures), on the midline, and 6.4 mm below the skull for the NH. By way of a midline approach, AdRSUß-gal solution (4x106 pfa/200 nL) was injected into the NH with a stainless steel 30-gauge cannula attached to a 10-µL Hamilton microsyringe by PE-10 polyethylene tubing. Central injections were delivered over the course of 10 minutes. The respective control animals were injected with 200 nL of saline–3% sucrose. The incisions were sutured, and the mice were returned to their cages after recovering from anesthesia.
Four days after direct injection of AdRSVß-gal into the NH, the mice were anesthetized and perfused with 2% paraformaldehyde in PBS, pH 7.4. Whole brains were excised and postfixed in the same solution. The brain sections were analyzed for ß-gal expression by histochemical analysis for cultured cells14 and tissues.15 Brain sections were rinsed twice for 10 minutes in a solution containing 2 mmol/L MgCl2 in 0.1 mol/L PBS. The sections were then incubated at 37°C in 5-bromo-4-chloro-3-indolyl-ß-D-galactoside (X-gal) in PBS for 2 hours. Finally, the sections were rinsed in PBS and photographed by light microscopy.
Data Analysis
Cell cultures were visualized under magnification (x200), and
neurons and pituicytes were identified on the basis of their
characteristic morphology and on previous studies of cells identified
by immunostaining techniques. The cells of the SON,
PVN, and NH cultures that expressed ß-gal or GFP were
counted over 4 randomly selected fields within the middle of each
coverslip. The time course of ß-gal or GFP expression was
analyzed by a 2-factor ANOVA with repeated measures, followed
by pairwise comparisons with Tukey's post hoc test. Student's
t tests for independent samples were used when appropriate.
Control cultures showing only zero values at all time points were not
included in the statistical analysis. The data obtained from
multiple coverslips are presented as mean±SEM for each
experimental condition. Differences between samples were considered
significant at P
0.05.
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Results |
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In the first analysis, we determined the relationship between viral concentration, gene expression, and viral cytotoxicity in dissociated and cultured SON, PVN, and NH cells by comparing the relative number of cells expressing ß-gal versus GFP. Figure 1 (top panels) shows typical viable pituicytes and PVN and SON neurons after 6 days in culture that expressed GFP after transfection with AdRSVgfp (2x107 pfu) 3 hours after tissue dissociation. Part of each sample was transfected with AdRSVß-gal (2x107 pfu) rather than with AdRSVgfp. The expression of ß-gal in stained pituicytes and in PVN and SON neurons is illustrated in Figure 1 (bottom panels). No fluorescence or blue staining was observed in cultured cells treated in vitro with volume-matched saline–3% sucrose vehicle.
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The relationship between viral concentration and cytotoxicity in cultured murine NH, PVN, and SON cells transfected in vitro with AdRSVß-gal or AdRSVgfp was also established in these experiments. Compared with vehicle-treated cultures, transfection of PVN and SON cultures with AdRSVß-gal 3 hours after dissociation at concentrations of 2x106 and 2x107 pfu did not significantly affect cell survival (see Figure 2, top panels). Higher viral concentrations (2x108 pfu) caused a marked progressive decrease in the number of PVN and SON cells surviving over time. In comparison with lower viral titers, a concentration of 2x108 pfu applied to the PVN and SON cultures resulted in cells that remained as small spherical soma, did not develop processes, and eventually became detached and floated in the media. In NH cultures, the total increase of pituicytes over time was similar between vehicle-treated cultures and those transfected with a low viral concentration of 2x106 pfu. In contrast, there was no increase in the number of pituicytes observed over time at viral concentrations of 2x107 pfu, and there was a substantial decrease in the number of pituicytes at the viral concentration of 2x108 pfu. Similar results were observed in NH, PVN, and SON cultures transfected with AdRSVgfp (data not shown). Taken together these findings indicate that viral concentrations higher than 2x107 pfu per well decrease NH, PVN, and SON cell viability.
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Figure 3 shows typical cultured pituicytes 6 days after dissociation and transfection with AdRSVß-gal at low (2x106 pfu) and intermediate (2x107 pfu) viral concentrations. Figure 2 (bottom panels) summarizes the relationship between viral concentration and ß-gal or GFP expression in the cultured PVN and SON cells described above that were AdRSVß-gal– or AdRSVgfp–transfected in vitro 3 hours after dissociation. As expected, we observed that the expression of ß-gal and GFP in NH, PVN, and SON cultured cells was related to the amount of virus; ie, the higher the viral concentration, the higher the percentage of GFP-positive or ß-gal–positive cells visualized in the culture. We observed that for the same viral concentration, the percentage of cells expressing ß-gal in stained cultures was significantly higher than those expressing GFP in living cultures. This suggests that visual detection of GFP expression in living cells by fluorescence emission is less reliable than using histochemical detection of ß-gal in fixed cells. For example, we observed 17±4% of GFP-positive pituicytes of living cultures transfected with AdRSVgfp (2x106 pfu) 4 days earlier compared with 58±4% of ß-gal–positive pituicytes from cultures also transfected 4 days before with AdRSVß-gal (2x106 pfu). Similar differences were observed in PVN and SON cultures.
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The data summarizing the relationship between viral concentration and cell viability (Figure 2, top panels) and between viral concentration and percentage of gene transduction clearly indicate that the optimal viral concentration for in vitro expression of ß-gal or GFP in murine NH, PVN, and SON cultured cells is 2x107 pfu.
We were also able to directly transfect the NH of adult mice with AdRSVß-gal by using stereotaxic approaches. In this study, the animals were killed 4 days after virus administration for detection of ß-gal expression with X-gal staining. Inoculated animals (n=5) did not show signs of poor health or brain inflammation. Figures 4C and 4D show a typical whole pituitary of a mouse transfected with AdRSVß-gal (4x106 pfu/200 nL) administered directly into the NH and a pituitary of a mouse injected with an equal volume of saline–3% sucrose vehicle. As illustrated in Figures 4A and 4B, we were able to detect the expression of ß-gal in coronal sections of the PVN and SON in mice with AdRSVß-gal injected into their NH. This indicates that AdRSVß-gal was retrogradely transported from the injection site to the soma of neurons located in the PVN and SON. The back-labeled soma in the PVN are located at the site in this nucleus where magnocellular neurons are present. Curiously, we consistently observed that coronal sections taken from mice with vehicle injections into the NH and from nonsurgery control mice showed some endogenous ß-gal expression (light blue staining) in the SON. Interestingly, this endogenous staining was not observed in the PVN (Figures 4A and 4B), and also no staining of this nature was observed in NH, PVN, and SON dissociated and cultured cells.
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Discussion |
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The purpose of the present studies was to define the optimal parameters that promote Ad-mediated gene transfer to cells of the hypothalamus-neurohypophyseal tract of mice. The major findings of the present study are as follows: (1) ß-Gal expression is detected in a higher percentage of cells than GFP expression in cultures of the NH, PVN, and SON that were transfected in vitro with AdRSVgfp or AdRSVß-gal, respectively. (2) ß-Gal and GFP are expressed in cultured cells as a function of concentration. (3) Regardless of the genetic marker used, Ad concentrations >2x107 pfu are cytotoxic. (4) A stereotaxic approach can be used to directly transfect the NH of adult mice with AdRSVß-gal. (5) AdRSVß-gal is retrogradely transported from the NH, and expressed ß-gal is present in the hypothalamic PVN and SON magnocellular neurons.
Virus-mediated gene delivery to the NH, PVN, and SON has significant potential for contributing to the further use of gene therapy to correct genetic disturbances in vasopressin production in PVN and SON magnocellular neurons and in diseases such as diabetes insipidus and possibly, hypertension. In our studies, we used replication-deficient Ad as a vector for gene delivery because it transfects postmitotic cells, can be used to transfect relatively large amounts of genetic material, and can be purified to high titers.2 16 17 In previous work, we demonstrated the feasibility of using Ad to deliver the gfp gene marker to subfornical organ (SFO) and SON neurons in rat pups.13 The present experiments were performed in the adult mouse. The mouse is a mammalian species widely used for genetic studies. Transgenic and gene-targeting technologies can be used in inbred mouse strains with defined genotypes. The neural transfection methods used here in the mouse are likely to provide an important methodological approach for physiological analysis of neural mechanisms implicated in pathological states. In this study, we overcame the obstacle of the small size of the mouse to reliably target the NH and hypothalamic magnocellular neurons in the PVN and SON.
We were able to transfect NH, PVN, and SON dissociated and cultured cells from adult mice with AdRSVgfp and AdRSVß-gal. The product of the Aequorea victoria gfp gene has the advantage that it can be visualized in living cells and is therefore very useful when characterizing the functional properties of target cells with imaging or electrophysiological methods.13 In contrast to GFP, E coli ß-gal requires that cells be fixed and that exogenous substrates or biological stains be used to identify cells containing transgenes.18 19 However, in the present study, we observed that at the same viral concentration, the expression of ß-gal was detected at levels almost 3 times greater than the expressed GFP. These results suggest that visualizing GFP fluorescence in living cells with commonly employed methods is likely to result in an underestimation of transfected cells.
In the experiments reported here, we defined the relationship between Ad concentration and the number of murine NH, PVN, and SON cells that survived in culture up to 8 days. Cultured cells treated with Ad concentrations >2x107 pfu did not survive as long as those treated with lower viral concentrations. This indicates that higher levels of Ad cause cytotoxic effects in cultured cells. Our results indicate that the viral concentration of 2x107 pfu appeared to be the optimal concentration because it resulted in high expression of GFP or ß-gal. This finding is in agreement with previous studies in which we demonstrated that SFO and SON dissociated and cultured cells from rat pups transfected with AdRSVgfp (2x106 to 107 pfu) showed a high percentage of stable GFP expression without affecting cell viability.13
An important feature of the Ad vector is that it can be taken up by axon terminals in the vicinity of the injection site and transported retrogradely to the cell soma. This has been demonstrated in the central nervous system of rats20 21 and mice.22 Previously, we took advantage of this to demonstrate in both rat pups13 and adults (E.C.V. et al, unpublished data, 1999) that SON injection of AdRSVgfp resulted in gene expression in SFO neurons. Although we were able to visualize GFP expression in dissociated, cultured SFO neurons from rat pups in which their SONs had been transfected with AdRSVgfp, we were not able to see GFP expression in coronal slices of either living or fixed tissues. In light of this problem, the lacZ gene was used in the present study as a marker. As illustrated in Figure 4, we were able to visualize the expression of ß-gal in fixed tissue at the pituitary injection site and in the SON and PVN. Although others have reported that Ad vectors can be used to transport genes to distant secondary targets in the murine brain,23 this is the first demonstration of Ad-mediated gene delivery from the NH to the soma of hypothalamic magnocellular PVN and SON neurons via retrograde transport. Although results from previous studies that we conducted in rats indicated that injections of Ad in concentrations comparable to those used in the present study did not cause an inflammatory response (E.C.V. et al, unpublished data, 1999), additional studies will be required to test whether this is also true for mice.
In summary, in these studies we have defined the optimal conditions for in vitro transfection with recombinant Ad of cells from the murine hypothalamic-neurohypophyseal system and have demonstrated that with conventional methods, it is easier to detect ß-gal expression than the presence of GFP. The stereotaxic approach employed here enabled us to selectively deliver genes to the magnocellular neurons of the PVN and SON at the hypothalamus via retrograde transport from their axon terminals in the NH of adult mice. Considering the pivotal role played by the hormone vasopressin in the control of body fluid volume and cardiovascular homeostasis, application of this experimental approach holds promise as a useful tool for studies of normal physiology and in pathological states related to genetic dysfunction of PVN and SON magnocellular neurons.
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Acknowledgments |
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The work was supported by grants from the National Institutes of Health (HL14388, HL57472, and DK54759), NASA (NAG5-6171), and the Office of Naval Research (N00014-97-1-0145). Elisardo C. Vasquez and Silvana S. Meyrelles were partially supported by CNPq-Brazil.
Received May 8, 1999; first decision July 1, 1999; accepted July 28, 1999.
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